Suitability of SSCP-PCR analysis for molecular detection of quinolone resistance in Campylobacter jejuni

Foodborne infections with Campylobacter spp. are increasing, especially antibiotic resistant strains are emerging. Quinolone resistant isolates can cause failure of therapy in severe clinical infections. Molecular characterisation is needed for the detection of resistant variants of C. jejuni. Therefore 23 isolates from poultry and human medicine as well as three control strains were tested for their minimal inhibitory concentration, their Single-Strand-Conformation-Polymorphism (SSCP)-PCR pattern (a method for the detection of resistance determining point mutations), and their sequence of the quinolone resistance determining region (QRDR). Six different SSCP types could be identified: two types for quinolone resistant isolates and other types containing so called silent mutations without influence on the resistance. A genotypic monitoring of the quinolone resistance in C. jejuni can be useful for the early detection of new resistance variants. As a screening method for detection of point mutations in the QRDR the SSCP-PCR can be applied. Compared to other genotypic methods the SSCP-PCR is less time and cost consuming and needs only standard technical equipment.     

Evaluation of the perioperative stress response in dogs administered medetomidine or acepromazine as part of the preanesthetic medication

OBJECTIVE: To compare the perioperative stress response in dogs administered medetomidine or acepromazine as part of the preanesthetic medication. ANIMALS: 42 client-owned dogs that underwent elective ovariohysterectomy. PROCEDURE: Each dog was randomly allocated to receive medetomidine and butorphanol tartrate (20 microgram/kg and 0.2 mg/kg, respectively, IM) or acepromazine maleate and butorphanol (0.05 and 0.2 mg/kg, respectively, IM) for preanesthetic medication. Approximately 80 minutes later, anesthesia was induced by administration of propofol and maintained by use of isoflurane in oxygen. Each dog was also given carprofen before surgery and buprenorphine after surgery. Plasma concentrations of epinephrine, norepinephrine, cortisol, and beta-endorphin were measured at various stages during the perioperative period. In addition, cardiovascular and clinical variables were monitored. RESULTS: Concentrations of epinephrine, norepinephrine, and cortisol were significantly lower for dogs administered medetomidine. Concentrations of beta-endorphin did not differ between the 2 groups. Heart rate was significantly lower and mean arterial blood pressure significantly higher in dogs administered medetomidine, compared with values for dogs administered acepromazine. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that for preanesthetic medications, medetomidine may offer some advantages over acepromazine with respect to the ability to decrease perioperative concentrations of stress-related hormones. In particular, the ability to provide stable plasma catecholamine concentrations may help to attenuate perioperative activation of the sympathetic nervous system.     

Laser ablation-combustion-GC-IRMS--a new method for online analysis of intra-annual variation of delta13C in tree rings

We present a new, rapid method for high-resolution online determination of delta13C in tree rings, combining laser ablation (LA), combustion (C), gas chromatography (GC) and isotope ratio mass spectrometry (IRMS) (LA-C-GC-IRMS). Sample material was extracted every 6 min with a UV-laser from a tree core, leaving 40-microm-wide holes. Ablated wood dust was combusted to CO2 at 700 degrees C, separated from other gases on a GC column and injected into an isotope ratio mass spectrometer after removal of water vapor. The measurements were calibrated against an internal and an external standard. The tree core remained intact and could be used for subsequent dendrochronological and dendrochemical analyses. Cores from two Scots pine trees (Pinus sylvestris spp. sibirica Lebed.) from central Siberia were sampled. Inter- and intra-annual patterns of delta13C in whole-wood and lignin-extracted cores were indistinguishable apart from a constant offset, suggesting that lignin extraction is unnecessary for our method. Comparison with the conventional method (microtome slicing, elemental analysis and IRMS) indicated high accuracy of the LA-C-GC-IRMS measurements. Patterns of delta13C along three parallel ablation lines on the same core showed high congruence. A conservative estimate of the precision was +/- 0.24 per thousand. Isotopic patterns of the two Scots pine trees were broadly similar, indicating a signal related to the forest stand's climate history. The maximum variation in delta13C over 22 years was about 5 per thousand, ranging from -27 to -22.3 per thousand. The most obvious pattern was a sharp decline in delta13C during latewood formation and a rapid increase with spring early growth. We conclude that the LA-C-GC-IRMS method will be useful in elucidating short-term climate effects on the delta13C signal in tree rings.     

Estradiol-17β-induced calcium uptake and resorption in juvenile rainbow trout,Oncorhynchus mykiss

Juvenile rainbow trout, Oncorhynchus mykiss, were injected with estradiol-17β (E₂) in order to study the source of extra calcium needed during vitellogenesis. E₂-treatment increased the calcium uptake from the external medium as well as calcium mobilization from muscle and scale. Judged by the increase in plasma protein-bound calcium levels, the E₂-induced increase in calcium uptake is an apparent over-mobilization of calcium, i.e., the calcium uptake of the fish is in excess of what is found bound to plasma proteins. As the calcium excretion and calcium space (calculated from free plasma calcium levels) were unaffected, the excess calcium is suggested to be incorporated into internal calcium stores. This implies that the systems regulating vitellogenesis and calcium balance are integrated on the mechanistic or endocrine level, and that E₂ causes calcium mobilization of a magnitude geared to the needs of the sexually maturing female.     

Assessment of EST- and genomic microsatellite markers for variety discrimination and genetic diversity studies in wheat

It is likely that in the near future sequence information from sequencing programmes and EST libraries will generate an abundance of genic microsatellite markers. This study is focused on the assessment of their likely impact and performance vis-à-vis their genomic counterparts. Microsatellites from two sources were used to assess the genetic diversity in 56 old and new varieties of bread wheat on the UK Recommended List. A set of 12 microsatellite markers generated from genomic libraries and 20 expressed sequence tag (EST)-derived microsatellites were used in the study, and the performance of both marker sets assessed. The EST-derived or genic microsatellites delivered fingerprints of superior quality, amplifying clear products with few stutter bands. Diversity levels as revealed bygenic microsatellites are similar to the few published results. The PIC values for the genic markers were generally lower than those calculated for the genomic microsatellites, though advantages of both marker classes for variety identification applications are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of pathogenic races ofPhytophthora fragariae Hickman in Germany

Summary The fungusPhytophthora fragariae Hickman is the causal agent of red stele disease in strawberry (Fragaria × ananassa), this being a major disease in many areas with cool and moist conditions. Success of resistance breeding can be nullified by the appearence of specific races of the fungus capable of overcoming the introduced resistance. In some countries (USA, UK, Japan and Canada) races were identified by using a differential set of strawberry cultivars. The absence of an international standard differential set and the use of different test methods make a comparison of the identified races difficult or even lead to contradictory results for one variety/race combination. The aim of this study was to obtain information about the spectrum of pathogenic races in Germany as a basic contribution to research on resistance breeding against the fungus. The susceptibility of different strawberry cultivars to German isolates ofP. fragariae was evaluated. The inoculation was done by modifying a method described by Milholland et al. (1989). Rootedin vitro plants, four weeks after transferring them to the soil, were used for the investigation. Inoculation was done with a zoospore suspension of defined concentration. Up to now three German isolates, G-1, G-2, and G-3, can be separated by their ability to infect and produce oospores in the roots of the strawberry cultivars ‘Senga Sengana’, either ‘Saladin’, ‘Redgauntlet’ or ‘Climax’ andFragaria chiloensis clone ‘YaquinaB’. These results confirm the existence of pathogenic races ofP. fragariae in Germany and should be taken into consideration for resistance breeding against the fungus.     

Manganese availability and microbial populations in the rhizosphere of wheat genotypes differing in tolerance to Mn deficiency

Plant genotypes differ in their capacity to grow in soils with low manganese (Mn) availability. The physiological mechanisms underlying differential tolerance to Mn deficiency are poorly understood. To study the relationship between Mn content in soil, plant genotypes, and rhizosphere microorganisms in differential Mn efficiency, two wheat (Triticum aestivum L.) cultivars, RAC891 (tolerant to Mn deficiency) and Yanac (sensitive), were grown in a Mn‐deficient soil to which 5, 10, 20 or 40 mg Mn kg^–1 were added. The shoot dry matter of both cultivars increased with increasing Mn addition to the soil. At all soil Mn fertilizer levels, the tolerant RAC891 had a greater shoot dry matter and a higher total shoot Mn uptake than the sensitive Yanac. The concentration of DTPA‐extractable Mn in the rhizosphere soil of RAC891 at Mn20 and Mn40 was slightly lower than in the rhizosphere of Yanac. The population density of culturable microorganisms in the rhizosphere soil was low (log 6.8–6.9 cfu (g soil)^–1) in both cultivars and neither Mn oxidation nor reduction were observed in vitro. To assess the non‐culturable fraction of the soil microbial community, the ribosomal intergenetic spacer region of the bacterial DNA in the rhizosphere soil was amplified (RISA) and separated in agarose gels. The RISA banding patterns of the bacterial rhizosphere communities changed markedly with increasing soil Mn level, but there were no differences between the wheat cultivars. The bacterial community structure in the rhizosphere was significantly correlated with the concentration of DPTA‐extractable Mn in the rhizosphere, fertilizer Mn level, shoot dry matter, and total shoot Mn uptake. The results obtained by RISA indicate that differential tolerance to Mn deficiency in wheat may not be related to changes in the composition of the bacterial community in the rhizosphere.