Phylogenetic position and nucleotide diversity of Fusarium oxysporum f. sp. vanillae worldwide based on translation elongation factor 1α sequences

Fusarium oxysporum f. sp. vanillae is considered the most important fungus affecting vanilla crops around the world, causing rot on vanilla roots and stems. Previous studies showed that the ability to infect vanilla plants is a polyphyletic trait among strains of the Fusarium oxysporum species complex (FOSC). The same studies proposed a single origin for F. oxysporum f. sp. vanillae isolates sampled from Mexico, the centre of origin and distribution of vanilla. The aim of this work was to test the hypothesis of the monophyletic origin of a wider sample of isolates of F. oxysporum f. sp. vanillae infecting Mexican vanilla and estimate nucleotide diversity of pathogen isolates from the main vanilla‐producing countries. Sequence data for the TEF1α gene from 106 isolates was assembled. The phylogenetic analyses suggest that some Mexican isolates of F. oxysporum f. sp. vanillae belong in two well‐supported clades, mixed with isolates from Madagascar, Indonesia, Réunion and Comoros. The phylogenetic position of other Indonesian and Mexican isolates is unresolved. Estimations of nucleotide diversity showed that the population from Mexico is genetically more diverse than the other three populations from Madagascar, Indonesia and Réunion. The results support a polyphyletic origin of vanilla‐infecting isolates of F. oxysporum worldwide, and also reject the proposition that Mexican isolates have a single origin. The phylogenetic optimizations over the strict consensus tree of the ability to infect vanilla plants suggest that pathogenic strains around the world are the product of multiple shifts of pathogenesis and dispersion events.     

Diet selection by calves facing pairs of nutritionally complementary foods

Experimental evidence suggests that ruminant animals are capable of selecting a nutritionally balanced diet (i.e. a diet appropriate to their metabolic needs) from imbalanced but complementary foods. We tested this hypothesis by offering calves different combinations of the same complementary foods. A 5 × 5 Latin square design was used to assess the effect on diet selection of different combinations (termed as food A and food B) of two complementary foods (alfalfa-grass hay and maize grain) offered in separate feed bunks. In Treatment 1, food A and food B had the same composition, 50.0% hay and 50.0% grain (T50:50). In the rest of the treatments, hay–grain combinations in foods A and B were, respectively: 62.5–37.5% and 37.5–62.5% (T63:37); 75.0–25.0% and 25.0–75.0% (T75:25); 87.5–12.5% and 12.5–87.5% (T88:12); and 100.0–0.0% and 0.0–100.0% (T100:0). Daily intake data were analysed through repeated measures analysis. Crude protein intake was similar across treatments (P = 0.28). An increasing tendency (P = 0.01) in food B intake (the food with more grain content) from T50:50 to T100:0, however, led into a decreasing tendency (P < 0.001) in the protein: energy ratio of the diet across treatments. Grain consumption in treatments T75:25, T88:12 and T100:0 was higher than expected, leading to protein levels in the diet below those required for a balanced diet (predicted vs. observed: 19.38 vs. 16.67, P = 0.004; 20.53 vs. 16.10, P = 0.001; and 20.23 vs. 16.79 g kgMW^? 1 d^? 1, P = 0.015, for T75:25, T88:12 and T100:0, respectively). Although calves were potentially able to select a balanced diet in all feeding treatments, they failed when offered high grain foods at choice (75% of maize grain or more). Evolved mechanisms to regulate macronutrient intake under natural conditions may fail to adequately operate in artificial environments involving high-energy density foods (e.g. maize grain).     

Ethanol/Water extraction combined with solid-phase extraction and solid-phase microextraction concentration for the determination of chlorophenols in cork stoppers

The appearance of 2,4,6-trichloroanisole (TCA) in cork stoppers is of great concern because it can cause off-flavors in bottled wine. To prevent this sensorial defect, there should not be any traces of 2,4,6-trichlorophenol (TCP), 2,3,4,6-tetrachlorophenol (TeCP), or pentachlorophenol (PCP) in the finished corks, because they are the direct precursors of TCA. In the course of this study two methodologies based upon an extraction with ethanol/water mixtures to determine the chlorophenolic content in cork matrices were developed. The cork extract is preconcentrated using both solid-phase extraction and solid-phase microextraction methodologies. The latter was optimized by applying a full two-level factorial design. Finally, spiked ground corks at nanogram per gram levels of each chlorophenol were analyzed under optimal conditions and by applying both procedures. The obtained results demonstrate that chlorophenols can be detected in corks contaminated at the nanogram per gram level and, thus, these approaches can be successfully applied as quality control measures in the cork industry.     

Computed tomographic retrograde positive contrast cystography and computed tomographic excretory urography characterization of a urinary bladder diverticulum in a dog

A one‐year‐old intact male German shepherd dog was referred with a 3‐month history of dysuria and pollakiuria. Physical examination revealed a large firm mass in the caudal abdomen. Findings from survey radiography, negative contrast cystography, computed tomographic (CT) retrograde positive contrast cystography, and CT excretory urography were consistent with a large urinary bladder diverticulum. An exploratory laparotomy revealed a normal wall appearance in the ventral compartment (true bladder) and marked thinning of the wall in the dorsal compartment (diverticulum). Both ureters inserted into the ventral compartment. The dorsal compartment was excised and histopathology confirmed the diagnosis of urinary bladder diverticulum.     

Internal standardization--atomic spectrometry and geographical pattern recognition techniques for the multielement analysis and classification of Catalonian red wines

Major and minor (K, P, Ca, Mg, Na, Fe, Mn, Zn, and Sr) and trace (Ba, Ni, Pb, V, Co, Cd, and Sb) elements from wine samples from the Denomination of Origin (DO) Empordà-Costa Brava (Catalonia, Spain) were analyzed by inductively coupled plasma atomic emission spectrometry (ICP-AES) and mass spectrometry (ICP-MS) respectively. Previously, a comparison of different calibration methodologies and sample digestion treatments had been carried out using ANOVA statistical tool. The obtained results demonstrated that internal standardization provides reliable results with the advantage that no further manipulation of the sample is needed. A principal component analysis of the concentration data was performed to differentiate the samples of DO Empordà-Costa Brava from wine samples from other wine-producing regions in Spain (i.e., Penedès, Somontano, and Rioja). It was found that Sr and Ba contents discriminate the two DO groups. Moreover, a discriminant analysis function involving both variables distinguishes the two groups with a 100% classification rate. At the level of the leave-one-out cross-validation, all of the Empordà-Costa Brava samples were well classified, whereas the other DOs presented two borderline misclassifications.     

Rapid methodology for antigenic profiling of FMDV field strains and for the control of identity, purity and viral integrity in commercial virus vaccines using monoclonal antibodies

Monoclonal antibodies (MAbs) developed against different foot-and-mouth disease virus (FMDV) vaccine strains were extensively used to study any possible antigenic variations during vaccine production in Argentine facilities. Additionally, a typing ELISA using strain specific MAbs was developed to detect potential cross contaminations among FMDV strains in master and working seeds with high specificity and sensitivity and to confirm strains identity in formulated vaccines. This assay was carried out for the South American strains currently in use in production facilities in Argentina (A24/Cruzeiro, A/Argentina/01, O1/Campos and C3/Indaial) and for the strain O/Taiwan, produced only for export to Asia. These non-cross reactive MAbs were also used to analyze the integrity of viral particles belonging to each one of the individual strains, following isolation of 140S virions by means of sucrose density gradients from the aqueous phase of commercial polyvalent vaccines. Antigenic profiles were defined for FMDV reference strains using panels of MAbs, and a coefficient of correlation of reactivity with these panels was calculated to establish consistent identity upon serial passages of master and production seeds. A comparison of vaccine and field strain antigenic profiles performed using coefficients of correlation allowed the rapid identification of two main groups of serotype A viruses collected during the last FMD epidemic in Argentina, whose reactivity matched closely to A/Argentina/2000 and A/Argentina/2001 strains.     

In vitro growth requirements for the fish pathogen Philasterides dicentrarchi (Ciliophora,Scuticociliatida)

Philasterides dicentrarchi is a scuticociliate causing fatal disease in farmed turbot and sea bass. In view of its high virulence and endoparasitic location, this parasite cannot be effectively controlled by formalin baths, and no systemic chemotherapeutic treatments have yet proved effective; immunoprophylaxis may thus be an attractive alternative approach. Since vaccine development is greatly facilitated by axenic culture of the pathogen, we have developed a simple axenic culture system based on commercially available Leibovitz L-15 medium, supplemented with fetal bovine serum, lipids (lecithin and Tween 80), nucleosides and glucose. After 1 week's culture under optimal conditions (salinity 10 per thousand, pH 7.2, temperature between 18 and 23 degrees C), yields of 1-2 x 10(5)cells/ml were obtained. Even cultures with seeding densities as low as 20 cells/ml were found to produce a good yield of ciliates (about 6 x 10(4)cells/ml) after 11 days of incubation. The ciliates thus obtained were free of contamination by other microorganisms, enabling preparation of pure P. dicentrarchi antigens for vaccine development studies.     

The effects of repeated applications of the molluscicide metaldehyde and the biocontrol nematode Phasmarhabditis hermaphrodita on molluscs, earthworms, nematodes, acarids and collembolans: a two-year study in north-west Spain

Over two years, six consecutive field experiments were done in which the chemical molluscicide metaldehyde and the nematode biocontrol agent Phasmarhabditis hermaphrodita (Schneider) were applied at the standard field rates to replicated mini-plots successively planted with lettuce, Brussels sprouts, leaf beet and cabbage, to compare the effectiveness of different treatments in reducing slug damage to the crops. Soil samples from each plot were taken prior to the start of the experiments, and then monthly, to assess the populations of slugs, snails, earthworms, nematodes, acarids and collembolans. The experiments were done on the same site and each plot received the same treatment in the six experiments. The six treatments were: (1) untreated controls, (2) metaldehyde pellets, (3 and 4) nematodes applied to the planted area 3 days prior to planting without or with previous application of cow manure slurry, (5) nematodes applied to the area surrounding the planted area 3 days prior to planting, and (6) nematodes applied to the planted area once (only in the first of the six consecutive experiments). Only the metaldehyde treatment and the nematodes applied to the planted area at the beginning of each experiment without previous application of manure significantly reduced slug damage to the plants, and only metaldehyde reduced the number of slugs contaminating the harvested plants. The numbers of slugs, snails and earthworms in soil samples were compared among the six treatments tested: with respect to the untreated controls, the numbers of Deroceras reticulatum (Müller) were significantly affected only in the metaldehyde plots, and the numbers of Arion ater L only in the plots treated with nematodes applied to the planted area 3 days prior to planting without previous application of manure; numbers of snails (Ponentina ponentina (Morelet) and Oxychilus helveticus (Blum)) were not affected by the treatment. The total numbers of all earthworm species and of Lumbricus spp were unaffected by the treatment, but Dendrobaena spp increased significantly in the plots treated with manure. The numbers of nematodes, acarids and collembolans in soil samples were compared between the untreated controls and the treatments with nematodes applied 3 days prior to planting to the planted area or to the surrounding area, without previous application of manure: the treatment had a significant effect on the number of nematodes in soil samples, but acarids and collembolans were unaffected.