Determination of strophanthidin in ingesta and plant material by LC-MS/MS

An LC-MS/MS method was developed for the semiquantitative determination of strophanthidin glycosides in ingesta from animals. Strophanthidin glycosides were simultaneously extracted and hydrolyzed to the strophanthidin aglycone using aqueous methanolic hydrochloric acid and the extracts cleaned up using solid-phase extraction. Extracts were analyzed using reverse-phase HPLC coupled with positive ion electrospray mass spectrometry. Characteristic product ion spectra were produced by fragmentation of the [M + H](+) precursor ion for each analyte. Quantitation was performed using the internal standard method with digitoxigenin serving as the internal standard. The method detection limit was calculated to be 0.075 microg/g, and the limit of quantitation was calculated to be 0.24 microg/g for strophanthidin in control rumen samples. This method was used in diagnostic investigations to confirm fatal strophanthidin glycoside poisonings in horses.     

Essential Oil Composition of Norwegian Peppermint (Mentha × piperita L.) and Sachalinmint [Mentha sachalinensis (Briq.) Kudô]

Production of varieties of the medicinal plants Mentha     

Field evaluation of treatments for the control of the bacterial apical necrosis of mango (Mangifera indica) caused by Pseudomonas syringae pv. syringae

Bacterial apical necrosis is a critical disease in the main production area of mango in Europe. It is caused by Pseudomonas syringae pv. syringae, and produces necrotic lesions on mango buds and leaves, causing severe yield losses due to a decrease of flowering and fruit set. A field study to evaluate control treatments against bacterial apical necrosis was carried out during three seasons on mango trees cv. Tommy Atkins in Huelva (Spain). Experimental treatments included Bordeaux mixture, fosetyl-Al, acibenzolar-s-methyl, gibberelic acid, silicon gel, a mixture between acibenzolar-S-methyl and Bordeaux mixture, and combined applications of fosetyl-Al with Bordeaux mixture or silicon gel. The treatments which caused a consistent reduction in bacterial apical necrosis symptoms at similar levels to the conventional treatment with Bordeaux mixture, were the plant resistance activator acibenzolar-S-methyl and the phosphonate derivative fosetyl-Al applied singly or in combination with other compounds, which could be alternative treatments. These treatments showed a significant decrease in the necrotic buds and/or leaves numbers; however, minor differences in P. syringae-like population levels were observed. The analysis of the inhibitory and bactericidal concentrations of cupric compounds against P. syringae strains isolated from mango tissues suggests that the commercial copper-based treatments with Bordeaux mixture used in the management of mango crops do not work in a bactericidal mode of action.     

Field resistance to QoI fungicides in Podosphaera fusca is not supported by typical mutations in the mitochondrial cytochrome b gene

BACKGROUND: A single nucleotide polymorphism in the mitochondrial cytochrome b gene confers resistance to strobilurin (QoI) fungicides in phytopathogenic fungi. Recent studies have revealed worrying levels of resistance to strobilurins in Podosphaera fusca (Fr.) U Braun & N Shishkoff comb. nov. [ = Sphaerothecafusca (Fr.) S Blumer], the main causal agent of cucurbit powdery mildew in Spain. In the present study the underlying resistance mechanism to QoI fungicides in the Spanish populations of P. fusca was investigated. RESULTS: Analysis of the Q(o) domains of cytochrome b in a collection of isolates revealed that none of the typical mutations conferring resistance to QoI, including the G143A and F129L substitutions, was present in the QoI-resistant isolates. Moreover, although different amino acid polymorphisms were observed in the two regions spanning the Q(o) site, none of them consistently distinguished QoI-resistant from QoI-sensitive strains. Exposure to salicylhydroxamic acid (SHAM), a specific inhibitor of alternative oxidase, in the presence of trifloxystrobin did not have any effect on QoI resistance, ruling out alternative respiration as the mechanism of resistance. Sensitivity tests to a battery of respiration inhibitors revealed high levels of cross-resistance to all Qo-inhibitors tested but not to Qi-inhibitors, these features resembling those of a target-site-based resistance. CONCLUSIONS: The results indicate that the mechanism responsible for QoI resistance in P. fusca is not linked to typical mutations in cytochrome b gene and that the absence of the G143A substitution cannot be explained by an intron following codon 143. These are important observations, especially in relation to the possible molecular diagnosis of resistance.     

Fresh-wood bending: linking the mechanical and growth properties of a Norway spruce stem

To provide data and methods for analyzing stem mechanics, we investigated bending, density and growth characteristics of 207 specimens of fresh wood from different heights and radial positions of the stem of one mature Norway spruce (Picea abies L. Karst.) tree. From the shape of each stress-strain curve, which was calculated from bending tests that accounted for shear deformation, we determined the modulus of elasticity (MOE), the modulus of rupture (MOR), the completeness of the material, an idealized stress-strain curve and the work involved in bending. In general, all mechanical properties increased with distance from the pith, with values in the ranges of 5.7-18 GPa for MOE, 23-90 MPa for MOR and 370-630 and 430-1100 kg m(-3) for dry and fresh wood densities, respectively. The first three properties generally decreased with stem height, whereas fresh wood density increased. Multiple regression equations were calculated, relating MOR, MOE and dry wood density to growth properties. We applied these equations to the growth of the entire stem and considered the annual rings as superimposed cylindrical shells, resulting in stem-section values of MOE, MOR and dry and fresh densities as a function of stem height and cambial age. The standing tree exhibits an inner stem structure that is well designed for bending, especially at a mature stage.     

Genetic Analyses for TSV-Susceptible and TSV-Resistant Pacific White Shrimp Litopenaeus vannamei using M1 Microsatellite

Allelic frequency differences at the highly polymorphic MI microsatellite of B20 locus were examined in Taura Syndrome Virus (TSV)-susceptible (Kona) and TSV-resistant (Select) lines of Pacific white shrimp Litopenaeus vannamei . DNA was screened from 1,009 cultured L. vannamei , 166 from the Kona line and 843 (97 broodstock and 746 offspring) from the Select line. A total of 34 different MI alleles were found, 41.2% of which were shared by the two lines, and 58.2% were unique to either line. Only seven (20.6%) of the 34 alleles had similar frequencies in the two lines. The most frequent (22.4%) allele in the Select line (218 bp) was absent from the Kona line. The most frequent (9.6%) allele in the Kona line (236 bp) was not observed in the Select line. Significant genetic differentiation between the two lines was shown using contingency table analysis (X²= 891.20, df = 33, P < 0.0001), the exact test ( P < 0.00001), and F_st, analysis (0.06). Comparison of MI allele frequencies between the TSVresistant Select and TSV-susceptible Kona lines and between the top 12.5% (Top Select) and the bottom 12.5% (Bottom Select) surviving families to TSV challenge in the Select line suggested associations of MI alleles with TSV resistance. These observations suggest testable hypotheses in future experiments aimed at finding genetic markers associated with resistance to TSV and markers that could be used for marker-assisted selection of broodstock.     

Prevalence and transmission of Neospora caninum within infected Swedish dairy herds

In this long-term study, the changes in within-herd Neospora caninum seroprevalences and the relative importance of horizontal and vertical parasite transmission in Swedish dairy herds were investigated. The within-herd prevalences varied between 5.8% and 65.0% when the herds were first sampled. Comparing the prevalences of year 2000 with those found 2-3 years later, nine out of 14 investigated herds had a similar or lower within-herd prevalence while five herds had an increased portion of seropositive individuals. Three herds were free or almost free from infection at their last sampling. Changes in prevalence were not related to the level of prevalence that each herd had when first sampled. Antibody avidity measurements revealed that the majority of seropositive individuals in all herds were chronically infected. Dam-calf pair investigations and inspection of genealogic trees supplemented with antibody test results showed that vertical transmission was by far the dominant transmission route in all herds. Taken together, these results indicate that the prevalence of N. caninum infection in Swedish dairy cattle can be successfully reduced if actions to eliminate infection in a herd are combined with preventive measures to avoid re-infection.     

Larval development assay for detection of anthelmintic resistance in cyathostomins of Swedish horses

The aim of this study was to investigate the suitability of a larval development assay (LDA) for the determination of anthelmintic resistance in cyathostomin nematode populations of the horse. In addition, comparison of results between geographic regions, types of horse establishment, and the use of anthelmintics in Sweden, was established. Seventy horse herds from different parts of Sweden were sampled, and strongyle eggs from the faeces of 54 of those were investigated by an LDA (DrenchRite). The following anthelmintics were tested: thiabendazole (TBZ), levamisole (LEV), ivermectin monosaccharide (IVM-MS), ivermectin aglycone (IVM-AG) and pyrantel (PYR). The LC50 values for TBZ and LEV were generally lower than those previously reported in other LDA studies on horse nematodes. This could be related to the infrequent use of these compounds for the past 20 years in Sweden. In this study, there was a great variation within and between assay plates that could not be explained. Still the LC50 values differed significantly between the regions for all anthelmintics, except for pyrantel. The highest LC50s were observed in parasite populations from the south of Sweden. There were no significant differences between riding schools and studs. Limitations of this technique exist, namely the lack of established cut-off values for susceptible and resistant populations and interpretation problems related to multi-species infections. Although there are advantages with LDA such as the possibility of testing several compounds simultaneously without interference with the deworming programmes on the farms, we conclude that LDA currently is not a reliable alternative to the faecal egg count reduction test (FECRT).     

Evaluation of gastro-intestinal nematode parasite control strategies for first-season grazing cattle in Sweden

A three-year grazing experiment (1998-2000) was conducted with first-season grazing cattle (FSGC) on improved pastures in central-eastern Sweden. Comparison was made between five groups with 10 calves in each group where four of these were set stocked and either (1) untreated, (2) ivermectin bolus treated, (3) subjected to biological control with the nematophagous fungus Duddingtonia flagrans, or (4) treated with a copper wire particle bolus. The fifth treatment was an evasive grazing strategy, whereby untreated calves were turned out onto pasture used by older cattle the previous year and then these calves were moved to silage aftermath in mid-July. To introduce low-levels of parasite infection to the experiment, each animal received a 'priming dose' of approximately 5,000 Ostertagia ostertagi and 5,000 Cooperia oncophora infective third stage larvae immediately prior to the start of the first grazing year of the trial.Results showed that efficient and sustainable parasite control of FSGC was possible to achieve without the use of anthelmintics by using turnout pastures that the previous year had been grazed by older cattle, in combination with a mid-July move to aftermath leys. Biological control also proved beneficial but the efficacy was impaired if high faecal egg counts coincided with rapid dung pat degradation due to heavy rainfall. No indication of parasite control was observed with the copper wire particle bolus. It was also demonstrated that the impact of gastrointestinal (GI) parasitism varied between years and that the level of overwintering contamination is important but likewise, is unpredictable. Although faecal egg counts in 1999 were low, due both to a delayed turnout and drought for the major part of the grazing season, deposited eggs successfully developed to infective larvae and overwintered in large numbers. The population of overwintered infective larvae at the time of turnout in early May played an important role in the course of infection in 2000 and resulted in an average 65 kg advantage of the ivermectin treated calves compared with the untreated calves.Thus, this three-year grazing experiment has emphasised the importance of subclinical gastrointestinal nematode infections in FSGC in Sweden. In addition, the study has shown that adequate parasite control may be achievable without the use of anthelmintics.