Perspectives on molecular detection methods of lyssaviruses

Lyssaviruses belong to the family Rhabdoviridae within the order Mononegavirales. Their genomes consist of a negative stranded linear RNA. Although dog transmitted rabies causes the majority of the estimated 55 000 worldwide fatalities per year, bat lyssaviruses have also caused human cases. Based on a broad range of different host species, their geographical distribution and the evolutionary age lyssaviruses display a high degree of genetic variability. Thus the development of molecular diagnostics for the reliable detection and identification of members of the genus Lyssavirus (pan-lyssavirus assay) is a challenge, but is valuable for the fight against rabies worldwide. In this study different published PCR-systems or primers for the detection of a wide range of lyssaviruses were tested as real-time RT-PCRs with different one-step and two-step assays. Comparison of different two-step assays by varying the RT-chemistry revealed differences in sensitivity. Nevertheless, most of the tested one-step systems provided an improved performance, including a reduced assay time and a reduced risk of cross-contaminations, when compared to the optimized two-step assay. Finally, we also provide an overview of additional state-of-the-art molecular methods to detect and differentiate lyssavirus species in general.     

Identification of differentially expressed protective genes in liver of two rainbow trout strains

Since 1975, the rainbow trout strain BORN (Germany) has been bred in brackish water from a coastal form imported from Denmark. Accompanying phenotypic monitoring of the adapted BORN trout until now revealed that this selection strain manifested a generally elevated resistance towards high stress and pathogenic challenge including lower susceptibility towards Aeromonas salmonicida infections in comparison to other trout strains in local aqua farms. We focus on the elucidation of both, genetic background and immunological basis for the increased survivorship to infections. A first comparison of gene expression profiles in liver tissue of healthy rainbow trout from the local selection strain BORN and imported trout using a GRASP 16K cDNA microarray revealed six differentially expressed genes evoking pathogen and wounding responses, LEAP2A (encoding for liver-expressed antimicrobial peptide), SERPINA1 (alpha-1 antitrypsin), FTH1 (middle subunit of ferritin), FGL2 (fibroleukin), CLEC4E (macrophage-inducible C-type lectin), and SERPINF2 (alpha-2 antiplasmin). Since the latter gene is not described in salmonid species so far, our first aim was to characterize the respective sequence in rainbow trout. Two trout SERPINF2 genes were identified, which share only 48% identical amino acid residues and a characteristic SERPIN domain. Second, we aimed to analyse the expression of those genes after temperature challenge (8 °C and 23 °C). Only FTH1 was upregulated in BORN and import trout after increase of temperature, while SERPINA1 and FGL2 were only elevated in import trout. Third, the expression of all named genes was analyzed after pathogen challenge with A. salmonicida subsp. salmonicida. As a main finding, we detected a comparably faster regeneration of LEAP2A mRNA abundance in BORN trout following bacterial infection. Ingenuity Pathways Analysis suggested a functional interplay among the mentioned factors and the pro-inflammatory cytokine TNF, whose stronger expression was validated in liver of BORN trout. This data indicate that the examined genes contribute to an improved first barrier against invading pathogens in BORN trout.     

Epizootic hemorrhagic disease virus infection of type I interferon receptor deficient mice

Type I interferon receptor deficient (IFNAR(-/-)) mice were infected with an Israeli isolate of serotype 7 of epizootic hemorrhagic disease virus (EHDV; Orbivirus, Reoviridae). Two out of two mice that received 5×10(5) 50% tissue culture infectious doses (TCID(50)) by intraperitoneal injection died or were euthanized in a moribund state on day 5 after infection. One mouse out of three that had been inoculated with 5×10(2) TCID(50) died on day 7 while the remaining mice did not show any clinical signs and survived until the end of the experiment. Spleens of all dead mice were highly positive in an EHDV real-time RT-PCR (quantification cycle values ≤15) and contained ≥10(5.8) TCID(50) of virus per ml of homogenate. The viral RNA content and virus titer in the spleens of the two surviving mice, on the other hand, were over 100-fold lower. Different from data reported for BTV, the outcome of EHDV infection of IFNAR(-/-) mice is dose-dependent and subclinical infections can occur.     

Development of a technique for continuous perineural blockade of the palmar nerves in the distal equine thoracic limb

Objective To develop a technique for placing continuous peripheral nerve block (CPNB) catheters adjacent to palmar nerves in horses and to evaluate the effect of low‐volume local anesthetic (LA) infusion on nociception in the distal equine thoracic limb. Study design In vitro and in vivo laboratory investigation. Study material and animals Forty‐two thoracic limbs from 22 equine cadavers and five horses. Methods Thoracic limb specimens were dissected to find landmarks for catheter insertion adjacent to medial and lateral palmar nerves. Based on the anatomy of the proximal metacarpus, a technique for placing palmar CPNB catheters was developed and the potential for catheter dislodgement studied in vitro by fluoroscopic visualization during passive carpal flexion and dye injection following simulated limb motion. The feasibility of CPNB catheter instrumentation in standing, sedated horses was tested in five animals, with ultrasound control. Electrical and mechanical stimulation thresholds and response latencies for hoof withdrawal responses (HWR) were determined following saline or LA infusion. Results Medial and lateral CPNB catheters were inserted percutaneously 2 and 4–5 cm, respectively, distal to the accessory carpal bone and advanced for ～7 and 10 cm, respectively, to place the tip just proximal to the communicating branch of the nerves. Catheters were placed correctly in 88% and 85% of cadaver limbs. In the standing horses, LA infusion not only increased HWR thresholds and latencies to noxious mechanical or electrical stimulation but also caused vasodilation and limb swelling over time. Conclusion The technique, developed in vitro, for placing and maintaining palmar CPNB catheters in the equine thoracic limb was successfully applied in vivo. Catheters were well tolerated but LA infusion may cause limb swelling, suggesting a need for further exploration of drug and infusion regimens. Clinical relevance Continuous perineural LA infusion along palmar nerves may develop into an effective analgesic technique in horses suffering from lower limb pain.     

Cation treatment and drying‐temperature effects on nonylphenol and phenanthrene sorption to a sandy soil

The objective of this study was to investigate the effects of mono‐ and polyvalent cations on sorption of the two hydrophobic compounds nonylphenol (NP) and phenanthrene (Phe). To this end, exchange sites of a sandy soil were saturated with either Na⁺, Ca²⁺, or Al³⁺ and excess salts were removed by washing. The samples were then sterilized and either stored moist, dried at room temperature, or at 20°C, 60°C, or 105°C in a vented oven. Saturation with Na⁺ led to an increase of dissolved organic C (DOC) concentration in the soil water extracts, whereas the polyvalent cations Ca²⁺ and Al³⁺ decreased it. The ¹H‐NMR relaxometry analyses showed that Al³⁺ restricted the mobility of water molecules that are confined within the SOM structure to a higher extent than Ca²⁺ or Na⁺. According to contact‐angle (CA) analyses, cation treatment did not significantly change the wetting properties of the samples. Batch sorption–desorption experiments showed no clear salt‐treatment effects on the sorption and desorption equilibria or kinetics of NP and Phe. Instead, the sorption coefficients and sorption hysteresis of NP and Phe increased in dry soil. With increasing drying temperature the CA of the soils and the sorption of both xenobiotics increased significantly. We conclude that structural modifications of SOM due to incorporation of polyvalent cations into the interphase structure do not modify the sorption characteristics of the soil for hydrophobic compounds. Instead, increasing hydrophobization of organic soil constituents due to heat treatment significantly increased the accessible sorption sites for nonpolar organic compounds in this soil.     

Potassium efficiency mechanisms of wheat,barley, and sugar beet grown on a K fixing soil under controlled conditions

Plant species differ in their potassium (K) efficiency, but the mechanisms are not clearly documented and understood. Therefore, K efficiency of spring wheat, spring barley, and sugar beet was studied under controlled conditions on a K fixing sandy clay loam. The effect of four K concentrations in soil solution ranging from low (5 and 20 μM K) to high (2.65 and 10 mM K) on plant growth and K uptake was investigated at 3 harvest dates (14, 21, and 31 days after sowing). The following parameters were determined: shoot dry matter (DM), K concentration in shoot dry matter, root length (RL), root length/shoot weight ratio (RSR), shoot growth rate/average root length ratio (GR_s/aRL), K influx, and soil solution K concentrations. Wheat proved to have a higher agronomic K efficiency than barley and sugar beet, indicated by a greater relative yield under K‐deficient conditions. As compared to both cereals, sugar beet was characterized by higher K concentrations in the shoot dry matter, only 30—50 % of the root length, 15—30 % of the RSR and a 3 to 6 times higher GR_s/aRL. This means that the shoot of sugar beet had a 3 to 6 times higher K demand per unit root length. Even at low K concentrations in the soil solution, sugar beet had a 7 to 10 times higher K influx than the cereals, indicating that sugar beet was more effective in removing low available soil K. Wheat and barley were characterized by slow shoot growth, low internal K requirement, i.e. high K utilization efficiency, and high RSR, resulting in a low K demand per unit root length. At low soil K concentrations, both cereals increased K influx with age, an indication of adaptation to K deficiency. The mechanism of this adaptation merits closer investigation. Model calculations were performed to estimate the K concentration difference between the bulk soil and the root surface (ΔC_L) needed to drive the measured K influx. For the two cereals, the calculated ΔC_L was smaller than the K concentration in the soil solution, but for sugar beet, ΔC_L was up to seven times higher. This indicates that sugar beet was able to mobilize K in the rhizosphere, but the mechanisms responsible for this mobilization remain to be studied.     

Investigating the potential for genetic improvement of nitrogen and phosphorus efficiency in a Swiss large white pig population using chemical analysis

Pig production contributes to environmental pollution through excretion of phosphorus and nitrogenous compounds. European pig production requires annual imports of currently 36 million tons of soya bean, because domestic plant protein sources often do not meet the required protein quality. Most of the mineral phosphate sources are also imported. It is therefore desirable to improve nutrient deposition efficiency through selective breeding, that is to realise similar growth rates and carcass compositions as currently achieved but with a lower intake of dietary crude protein or phosphate. For a preliminary evaluation of the potential of selecting for increased nutrient deposition efficiency, we estimated genetic parameters for nitrogen and phosphorus efficiencies in a Swiss Large White pig population including 294 individuals. Nutrient efficiency phenotypes were obtained from wet-chemistry analyses of pigs of various live weights. Heritability of nitrogen efficiency was estimated at 41%. Heritability of phosphorus efficiency was very low (0.3%), but positive genetic correlations with nitrogen efficiency suggest that breeding for nitrogen efficiency would positively affect phosphorus efficiency. Further studies are needed to improve the quality of estimates and to obtain accurate high-throughput measures of nutrient efficiency to be implemented on farms.     

Substituted 3-furamides with specific activity against basidiomycetes

Substituted furanilides and furamides show very good activity against Basidiomycetes. Whilst N-cyclohexyl-x-methyl-3-furamides (where x- is one or more of the vacant positions in the furan ring) further substituted on the amide nitrogen have specific activity against Basidiomycetes (e.g. Ustilago nuda, U. scitaminea, Tilletia tritici and Rhizoctonia solani), N-cyclohexyl-N-methoxy-2,5-dimethyl-3-furamide also gives good control of other classes of fungi (e.g. Fusarium nivale and Helminthosporium gramineum). This compound is to be developed under the Code Number BAS 389 F as a mercury-free seed dressing alone or in combination with other fungicides.