A duplex real-time polymerase chain reaction assay for the detection of and differentiation between Dirofilaria immitis and Dirofilaria repens in dogs and mosquitoes

This study describes a duplex real-time polymerase chain reaction (PCR) assay for the detection and differentiation between Dirofilaria immitis and Dirofilaria repens in dog blood and mosquitoes. Regions of a cytochrome oxidase 1 (cox1) mitochondrial DNA fragment and the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA were amplified from microfilariae and adult worm samples, using a sensitive SsoFast? EvaGreen(?) based real-time PCR method coupled with melting-curve analysis. The limit of the real-time PCR in detecting microfilaria and adult worm DNA was also tested both in dog blood and in artificially infected microfilarial. Two peaks at different melting temperatures (T(m)) for D. immitis (mean ± SD=75.7 ± 0.3°C) and D. repens (mean ± SD=70 ± 0.7°C), respectively, were obtained for microfilarial and adult positive controls of both species when examined separately and together. The real-time PCR protocol was also efficient in detecting microfilarial and adult DNA of both species when tested in samples spiked with DNA from Aedes albopictus, in Aedes aegypti experimentally infected by D. repens and in Culex pipiens naturally infected by D. repens and D. immitis. The high sensitivity of real-time PCR confirmed its reliability in detecting small amounts of genomic DNA either in dog blood or mosquitoes (2.5 pg/μl and 3 × 10(-1)pg/μl for D. immitis and D. repens, respectively). This assay is proposed as a tool for the epidemiological surveillance of the two most important Dirofilaria species in areas where they are endemic and sympatric.     

Does afforestation increase bird nest predation risk in surrounding farmland?

Afforestation of agricultural land is increasingly used to deliver environmental benefits, but their effects on biodiversity remain poorly understood. This paper tests the hypothesis that afforestation changes predation processes in surrounding farmland, examining how the characteristics and landscape context of forest plantations affect predator (birds and mammalian carnivores) and key prey (rabbits and hares) abundances, and bird nest predation rates in Iberian cereal-steppes. Lagomorphs and predators were surveyed in fallow fields around 50 forest plantations, where predation rates were estimated using artificial nests set at 0, 100, 200 and 300 m from the forest edge. Recent plantations structurally similar to sparse (oak) or dense (pine) shrublands were associated with the highest hare and rabbit abundances, respectively, whereas both species avoided landscapes with high eucalyptus cover. In contrast, mature eucalyptus plantations showed strong positive effects on typical nest predators such as corvids and carnivores. Open farmland fragmentation favoured the abundance of lagomorphs and carnivores. Despite these effects and the high predation rate on artificial nests (49%), there was neither evidence for increased predation near plantation edges nor higher predation in fields with more lagomorphs and predators. However, predation tended to increase with cover by young oak plantations and overall forest plantation cover, to decrease with eucalyptus cover at both the local and landscape scales, and to peak in landscapes with intermediate edge densities. These results suggest that afforestation may have strong effects on bird nest predation rates by changing landscape composition and configuration, rather than by inducing local increases in predator and prey populations. Nevertheless, increased abundances of generalist predators associated with forest plantations may still be considered of conservation concern, thus supporting the recommendation for strongly restricting afforestation in areas important for open grassland birds. Where this is unavoidable, monitoring should be undertaken to provide early signals for bird population declines associated with predator increases, eventually triggering conservation action such as predator exclusion or removal.     

A new dihydroxysterol from the marine phytoplankton Diacronema sp

Diacronema sp. was cultured and its sterols were separated by column chromatography on silica gel. The new sterol 24-ethyl-4alpha-methyl-cholestane-3,20-diol (1) was characterised by NMR and MS spectrometry, as well as (22E)-24-ethyl-4alpha-methyl-5alpha-cholest-22-en-3beta-ol (2) and beta-sitosterol, the major components of the sterol fractions. Neither the biosynthetic origin of the new dihydroxysterol nor its role in the biochemistry of Diacronema is known.     

Irrigation of paddy soil with industrial landfill leachate: impacts in rice productivity,plant nutrition,and chemical characteristics of soil

Treated industrial effluents have high levels of nutrients and dissolved organic matter. The irrigation of rice by flooding can increase nutrient uptake and grain yield. Therefore, this study evaluated the nutrient contents in the shoots and grain of the rice crop and also the chemical of the soil after irrigation of the crop with leachate of the treated industrial effluent. A greenhouse experiment was conducted using pots filled with 20 kg of soil in a randomized block design with three replications. The treatments consisted of control (irrigation with distilled water) and four concentrations of the leachate (25, 50, 75, and 100 %) for irrigation. At the end of the experiment, the nutrient contents in tissues of rice plants, sterility of spikelets, and grain mass were evaluated. Results showed that irrigation with the leachate at 25 % content increased the macro- and micronutrients’ concentrations in the shoot biomass and grain, except for potassium and iron. Irrigation with the industrial leachate decreased tillering and grain yield; however, it increased chlorophyll content, sterility of spikelets, and sodium intake at this leachate concentration. The potassium and sodium levels and the electrical conductivity values of soils irrigated with treated industrial leachate were increased. The use of the treated leachate from industrial effluents is an alternative that reuses the nutritional load, but the volume of leachate should be limited and monitored to prevent the sodicity in the soil and problemsdue to eutrophication.     

Iron deficiency and pseudo-iron deficiency in hospitalized horses

Values for serum ferritin, serum iron, and serum total-iron binding capacity were determined in 100 hospitalized horses. Six foals had iron deficiency, as indicated by low serum ferritin and iron concentrations. Twenty-eight horses had low serum iron content and normal or high serum ferritin content and were considered to have pseudo-iron deficiency, possibly in response to infection. Ten horses had serum ferritin concentrations that were quite high, probably indicating iron overload.     

Xylogenesis of Pinus pinaster under a Mediterranean climate

Context

The knowledge on cambial activity in water-limited environments, such as the Mediterranean, is still fragmentary. Dendrochronological studies have determined that spring precipitation plays an important part in determining tree-ring width and the properties of tracheids. However, the complex relation between cambial phenology and climate is still far from understood.

Aims

We studied the influence of climate, especially water stress, on maritime pine wood formation with the aim of determining the influence of drought on cambial activity.

Methods

A plantation of maritime pine (Pinus pinaster) was selected in the west coast of Portugal, to monitor cambial activity and wood formation using anatomical observations and band dendrometers. The trees were monitored weekly over 2 years (2010 and 2011).

Results

Xylem differentiation started earlier when warmer late winter temperatures were observed. Water stress triggered an earlier stop of wood formation and also the formation of tracheids with smaller lumen area. In both years a bimodal pattern of stem radial increment was registered by band dendrometers with two periods of increment: one in spring and another in autumn. The xylem anatomy study suggests that the autumnal increment period corresponded mostly to stem rehydration, since the differentiation of new xylem cells by the cambium was not observed.

Conclusion

Maritime pine cambial activity appears to be under a double climatic control: temperature influences cambial onset and water availability growth cessation.     

Induction of systemic and local basophil and eosinophil responses in guinea pigs by the feeding of the tsetse fly Glossina morsitans

Guinea pigs infested with Glossina morsitans weekly for 5 weeks exhibited marked peripheral blood basophil and eosinophil responses to each infestation, with a dominant cutaneous basophil response to challenge infestation. G. morsitans feeding was completed within 3--10 min, depending upon prior exposure, and flies were reluctant to feed and probed longer on hyperexposed animals. Blood basophil responses exhibited the greatest increases over controls (up to 12-fold) compared to eosinophils (up to 3-fold). After the first and third infestations, both basophil and eosinophil levels increased, whereas after the second and fourth infestations both cell types declined. Greatest blood basophil responses developed after the first infestation with levels ranging from 0 to 14 +/- 9 cells/mm3 in infested animals to 0 and 2 +/- 2 cells/mm3 in uninfested controls. Eosinophilia increased with each infestation where levels ranged from 57 +/- 23 cells/mm3 after the first tsetse feeding to 110 +/- 20 cells/mm3 after the fourth infestation; compared to 11 +/- 11 to 50 +/- 12 cells/mm3 in uninfested controls. Fly-feeding sites were marked by hemorrhages, and probing behavior resulted in a line of small hemorrhages when the underside of the skin was examined. Histologically, G. morsitans feeding sites in naive guinea pigs 24 h post-infestation were dominated by mononuclear cells (93% of the infiltrate) with a weak granulocyte component, of which eosinophils were dominant (1.3%). Tsetse feeding sites in guinea pigs exposed 3 times previously were again dominated by mononuclear cells (57% of the infiltrate), but granulocytes comprised a significant part of the response (43% of the infiltrate) where basophils were dominant (25%).