The innate antiviral immune system of the cat: molecular tools for the measurement of its state of activation

The innate immune system plays a central role in host defence against viruses. While many studies portray mechanisms in early antiviral immune responses of humans and mice, much remains to be discovered about these mechanisms in the cat. With the objective of shedding light on early host-virus interactions in felids, we have developed 12 real-time TaqMan(?) qPCR systems for feline genes relevant to innate responses to viral infection, including those encoding for various IFNα and IFNω subtypes, IFNβ, intracellular antiviral factor Mx, NK cell stimulator IL-15 and effectors perforin and granzyme B, as well as Toll-like receptors (TLRs) 3 and 8. Using these newly developed assays and others previously described, we measured the relative expression of selected markers at early time points after viral infection in vitro and in vivo. Feline embryonic fibroblasts (FEA) inoculated with feline leukemia virus (FeLV) indicated peak levels of IFNα, IFNβ and Mx expression already 6h after infection. In contrast, Crandell-Rees feline kidney (CrFK) cells inoculated with feline herpes virus (FHV) responded to infection with high levels of IFNα and IFNβ only after 24h, and no induction of Mx could be detected. In feline PBMCs challenged in vitro with feline immunodeficiency virus (FIV), maximal expression levels of IFNα, β and ω subtype genes as well as IL-15 and TLRs 3, 7 and 8 were measured between 12 and 24h after infection, whereas expression levels of proinflammatory cytokine gene IL-6 were consistently downregulated until 48h post inoculation. A marginal upregulation of granzyme B was also observed within 3h after infection. In an in vivo experiment, cats challenged with FIV exhibited a 2.4-fold increase in IFNα expression in blood 1 week post infection. We furthermore demonstrate the possibility of stimulating feline immune cells in vitro with various immune response modifiers (IRMs) already known for their immunostimulatory properties in mice and humans, namely Poly IC, Resiquimod (R-848) and dSLIM?, a synthetic oligonucleotide containing several unmethylated CpG motifs. Stimulation of feline PBMCs with dSLIM? and R-848 effectively enhanced expression of IFNα within 12h by factors of 6 and 12, respectively, and Poly IC induced an increase in Mx mRNA expression of 28-fold. Altogether, we describe new molecular tools and their successful use for the characterization of innate immune responses against viruses in the cat and provide evidence that feline cells can be stimulated by synthetic molecules to enhance their antiviral defence mechanisms.     

Qualitative aspects in the analysis of pesticide residues in fruits and vegetables using fast, low-pressure gas chromatography-time-of-flight mass spectrometry

Quantitative method validation is a well-established process to demonstrate trueness and precision of the results with a given method. However, an assessment of qualitative results is also an important need to estimate selectivity and devise criteria for chemical identification when using the method, particularly for mass spectrometric analysis. For multianalyte analysis, automatic instrument software is commonly used to make initial qualitative identifications of the target analytes by comparison of their mass spectra against a database library. Especially at low residue levels in complex matrices, manual checking of results is typically needed to correct the peak assignments and integration errors, which is very time-consuming. Low-pressure gas chromatography-mass spectrometry (LP-GC-MS) has been demonstrated to increase the speed of analysis for GC-amenable residues in various foods and provide more advantages over the traditional GC-MS approach. LP-GC-MS on a time-of-flight (ToF) instrument was used, which provided high sample throughput with <10 min analysis time. The method had already been validated to be acceptable quantitatively for nearly 150 pesticides, and in this study of qualitative performance, 90 samples in total of strawberry, tomato, potato, orange, and lettuce extracts from the QuEChERS sample preparation approach were analyzed. The extracts were randomly spiked with different pesticides at different levels, both unknown to the analyst, in the different matrices. Automated software evaluation was compared with human assessments in terms of false-positive and -negative results. Among the 13590 possible permutations with 696 blind additions made, the automated software approach yielded 1.2% false presumptive positives with 23% false negatives, whereas the analyst achieved 0.8% false presumptive positives and 17% false negatives for the same analytical data files. False negatives frequently occurred due to challenges at the lowest concentrations, but 70% of them involved certain pesticides that degraded (e.g., captafol, folpet) or otherwise could not be detected. The false-negative rate was reduced to 5-10% if the problematic analytes were excluded. Despite its somewhat better performance in this study, the analyst approach was extremely time-consuming and would not be practical in high sample throughput applications for so many analytes in complicated matrices.     

Der einfluss Klimatischer Faktoren auf die Ertragsbildung im ISDV‐Standort vergleich Grüppenbühren‐Rauischholzhausen‐Puch

Im Standortvergleich zwischen den Standorten Grüppenbühren bei Oldenburg, Rauischholzhausen bei Gießen und Puch bei München werden in der 1. Mitteilung Zusammenhänge zwischen mittleren Trockenmasseerträgen bei Winterweizen einerseits und den Klima‐Elementen Temperatur, Niederschlag und Strahlungsbilanz andererseits aufgezeigt. Die der Auswertung zugrundeliegenden Daten wurden innerhalb der "Internationalen Stickstoff‐Dauerdüngungs‐ Versuche ISDV”; ermittelt. Die Versuchsreihe ISDV wurde auf insgesamt 24 Standorten in Europa als Stickstoffsteigerungsversuch (6 N‐Stufen ohne und 3 N‐Stufen mit organischer Düngung) mit einer dreigliedrigen Fruchtfolge in den Jahren 1972–1983 von der Internationalen Arbeitsgemeinschaft für Bodenfruchtbarkeit innerhalb der Internationalen Bodenkundlichen Union konzipiert und betreut. Die Ergebnisse lassen sich wie folgt zusammenfassen:

Die Ergebnisse lassen sich wie folgt zusammenfassen:

? Der Einfluss der Klima‐Elemente auf die untersuchten Ertragskomponenten differiert deutlich zwischen den Standorten.

? Eine unterschiedliche Wirksamkeit in den N‐Düngungsvarianten konnte am Standort Grüppenbühren und Rauischholzhausen in der Tendenz aufgezeigt werden.

? Für den Standort Puch war kein gesicherter Einfluss festzustellen.

? Weitere Auswertungsschritte auf der Ebene der Monatsdaten und Pentadenwerte sind vorgesehen.     

Effects of Soil Conditions on the Prevalence of Netted Scab

Abstract

In two field trial series different planting techniques, used to alter soil conditions at planting time, and rates of seed infection with netted scab were compared regarding their effects on plant development, yield, tuber size distribution and the prevalence of netted scab in the progeny. A greenhouse pot experiment was also carried out to study the influence of soil contamination and infection of seed with the netted scab organism on the prevalence of netted scab in the progeny. Both seed infection and planting technique affected the progeny tuber infection with netted scab. Seed infection also negatively influenced plant development and yield. It is concluded that seed infection affects plant development and yield more than soil contamination does, whereas soil contamination is more an important source of progeny tuber infection.     

Importance of bank erosion for sediment input, storage and export at the catchment scale

Purpose

The importance of bank erosion was quantified during three periods (October 2006–April 2007, May 2007–April 2008 and May 2008–April 2009) in the 486 km² catchment area of River Odense, Denmark. A catchment sediment budget was established including other sediment sources such as tile drains and surface runoff, in-channel and overbank sinks and storage and the resulting bed load and suspended sediment load exported from the catchment.

Material and methods

Bank erosion and sedimentation were measured using ca. 3,000 erosion pins established in 180 pin plots, each consisting of three vertical lines of pins. Thirty-six representative reaches, each with a length of 100 m, were selected by a stratified random procedure in GIS. Bed load and suspended sediment export from the catchment were measured using a bed load sampler and from continuous measurements of turbidity at the outlet gauging station.

Results and discussion

The gross sediment input from bank erosion during the three study periods amounted to 21,100–25,200 t in the River Odense catchment, which is considerably higher than the estimated input of sediment from tile drains and surface runoff, which amounted to 220–500 t and 0–100 t, respectively. The measured bed load (20–490 t) was five to 60 times lower than the suspended sediment export from the catchment (1,240–2,620 t) during the three study periods, with the largest difference occurring in the driest year. Sediment sinks and storage were of high importance for the catchment sediment budget as the measured in-channel storage of sediment on stream banks was as high as 16,200–20,100 t, and the overbank sediment sink was estimated at 360–3,100 t.

Conclusions

Bank erosion was the dominant sediment source (90–94 %) in the River Odense catchment during the three study years. In-channel and overbank sediment sinks and storage dominated the sediment budget as 79–94 % of the sediment input from all sources was not exported from the catchment during the three study years. Such a large attenuation of sediment in river channels and on floodplains is extremely important for fluvial habitats and ecology. Moreover, it has strong implications for attempts to document changes in sediment export following implementation of mitigation measures.