Cryopreservation of carnation (Dianthus caryophyllus L.) shoot tips by encapsulation-vitrification

Shoot tips excised from in vitro cultured plants of Dianthus caryophyllus L. (cv. Pallas, cv. Pink Candy and cv. Wanessa) were successfully cryopreserved using an encapsulation-vitrification method. Shoot tips (2–3 mm in length) were encapsulated in sodium alginate, precultured on liquid Murashige and Skoog (1962) medium supplemented with various sucrose concentrations (0.25, 0.5, 0.75, 1.0 M) for 24 h or 48 h and dehydrated with the vitrification solution PVS2 (up to 4 h) at 24 °C or 0 °C prior to direct immersion in liquid nitrogen (−196 °C). A maximum of shoot regeneration from cryopreserved shoot tips was obtained with the following combinations: preculture in 0.5 M sucrose and 180 min dehydration treatment at 0 °C for cv. Pallas (60% shoot formation), or preculture in 0.75 M and 200 min dehydration at the same temperature for cv. Pink Candy (66.6% shoot formation) and cv. Wanessa (73% shoot formation).     

Soil macroinvertebrate fauna of a Mediterranean arid system: Composition and temporal changes in the assemblage

Temporal variability is a key factor to understand the structure of belowground communities. Seasonal and annual variations are especially relevant in unpredictable desert ecosystems, where macroinvertebrates are poorly known, despite constituting an important group of soil organisms. In the present study, we analyse the composition and temporal (seasonal and annual) variations of soil macroinvertebrates in an arid area of southern Spain. During two years, macroinvertebrates were sampled in litter and belowground levels by means of soil cores. Results show that the assemblage was dominated by arthropods, especially Formicidae and Coleoptera. The assemblage differed between litter and belowground levels. In litter, detritivores dominated the community, while belowground fauna showed a similar proportion of detritivores and herbivores and a low percentage of predators. Litter and belowground assemblages showed seasonal variations in richness, abundance, biomass and composition, although variations were more marked in litter than belowground. Patterns of seasonal variation also differed between the two study years for both litter and belowground invertebrates. The seasonal and annual variability of the assemblage has potentially important implications for community dynamics in the study system, since the changes in species composition and trophic structure of soil invertebrate assemblages may affect species interactions and food web dynamics over time. Therefore, integrating temporal variability is likely to be crucial to understand soil community dynamics and food webs, especially in heterogeneous, variable systems as deserts.     

Protected wading bird species threaten relict centenarian cork oaks in a Mediterranean Biosphere Reserve: A conservation management conflict

Conservation management conflicts frequently arise when an overpopulation of a protected organism has negative effects on other valuable elements in the same ecosystem. We studied the interactions between a colony of protected tree-nesting wading birds and a remnant population of centenarian cork oaks that was part of the formerly dominant forests in the Doñana Biological Reserve (SW Spain). A significant increase in the tree mortality rates has been recorded in areas that are yearly influenced by the bird colony.We analysed a cohort of surviving trees using a gradient of nesting bird influence. Tree-nesting history, bird isotopic signature (δ¹⁵N), tree health-related parameters (defoliation, δ¹³C and leaf surface coverage by faeces) and several soil variables were evaluated. Bird influence was related to increased soil salinity. This increase correlated to increased water-use efficiency for the leaves and to crown defoliation, suggesting that the heavily occupied trees are under higher stress and in poorer health condition than the unoccupied ones. We tested structural equations models (SEM) that were based on hypothesised bird effects on the health of the trees. Soil-mediated effects of the nesting birds best explained the symptoms of the declining health of the trees, whereas the percent of leaves’ surface that was covered by faeces did not improve the fitted SEM model.For the reserve’s managers, a challenging trade-off exists between preserving the relict trees, which have a high genetic diversity and a key ecological role in these savannah-like ecosystems, and maintaining the current nesting area for these protected, but expanding, wading birds.     

Oxidant-antioxidant imbalance in horses infected with equine infectious anaemia virus

This study assesses the impact of equine infectious anaemia virus (EIAV) infection on the oxidant/antioxidant equilibrium of horses. Blood samples from 96 Romanian horses aged 1-25 years, were divided into different groups according to their EIAV-infection status, age, and time post-seroconversion. The effect of infection on oxidative stress was estimated by measuring enzymatic antioxidants (superoxide dismutase [SOD], glutathione peroxidase [GPx] and catalase), non-enzymatic antioxidants (uric acid and carotenoids), and lipid peroxidation (malondialdehyde [MDA]). Infection modified the oxidant/antioxidant equilibrium in the horses, influencing GPx and uric acid levels (P<0.05). Time post-seroconversion also contributed to oxidative stress imbalance, exhibiting a significant influence on both SOD and MDA concentrations in the blood (P<0.05). Animal age did not have a significant influence on oxidative stress. Recently infected horses (<1 year following seroconversion), and horses >5 years old, represented the most vulnerable category in terms of oxidative stress, followed by recently infected animals <5 years old. The results of this study are novel in implicating EIAV infection in the development of oxidative stress in horses.     

Climate change effects on trematodiases,with emphasis on zoonotic fascioliasis and schistosomiasis

The capacity of climatic conditions to modulate the extent and intensity of parasitism is well known since long ago. Concerning helminths, among the numerous environmental modifications giving rise to changes in infections, climate variables appear as those showing a greater influence, so that climate change may be expected to have an important impact on the diseases they cause. However, the confirmation of the impact of climate change on helminthiases has been reached very recently. Only shortly before, helminthiases were still noted as infectious diseases scarcely affected by climate change, when compared to diseases caused by microorganisms in general (viruses, bacteriae, protozoans). The aim of the present paper is to review the impact of climate change on helminthiases transmitted by snails, invertebrates which are pronouncedly affected by meteorological factors, by focusing on trematodiases. First, the knowledge on the effects of climate change on trematodiases in general is reviewed, including aspects such as influence of temperature on cercarial output, cercarial production variability in trematode species, influences of magnitude of cercarial production and snail host size, cercarial quality, duration of cercarial production increase and host mortality, influence of latitude, and global-warming-induced impact of trematodes. Secondly, important zoonotic diseases such as fascioliasis, schistosomiasis and cercarial dermatitis are analysed from the point of view of their relationships with meteorological factors. Emphasis is given to data which indicate that climate change influences the characteristics of these trematodiases in concrete areas where these diseases are emerging in recent years. The present review shows that trematodes, similarly as other helminths presenting larval stages living freely in the environment and/or larval stages parasitic in invertebrates easily affected by climate change as arthropods and molluscs as intermediate hosts, may be largely more susceptible to climate change impact than those helminths in whose life cycle such phases are absent or reduced to a minimum. Although helminths also appear to be affected by climate change, their main difference with microparasites lies on the usually longer life cycles of helminths, with longer generation times, slower population growth rates and longer time period needed for the response in the definitive host to become evident. Consequently, after a pronounced climate change in a local area, modifications in helminth populations need more time to be obvious or detectable than modifications in microparasite populations. Similarly, the relation of changes in a helminthiasis with climatic factor changes, as extreme events elapsed relatively long time ago, may be overlooked if not concretely searched for. All indicates that this phenomenon has been the reason for previous analyses to conclude that helminthiases do not constitute priority targets in climate change impact studies.     

Improved isolation procedure for azaspiracids from shellfish, structural elucidation of azaspiracid-6, and stability studies

Azaspiracids are a group of lipophilic polyether toxins produced by the small dinoflagellate Azadinium spinosum. They may accumulate in shellfish and can result in illnesses when consumed by humans. Research into analytical methods, chemistry, metabolism, and toxicology of azaspiracids has been severely constrained by the scarcity of high-purity azaspiracids. Consequently, since their discovery in 1995, considerable efforts have been made to develop methods for the isolation of azaspiracids in sufficient amounts and purities for toxicological studies, in addition to the preparation of standard reference materials. A seven-step procedure was improved for the isolation of azaspiracids-1-3 (1, 2, and 3) increasing recoveries 2-fold as compared to previous methods and leading to isolation of sufficiently purified azaspiracid-6 (6) for structural determination by NMR spectroscopy. The procedure, which involved a series of partitioning and column chromatography steps, was performed on 500 g of Mytilus edulis hepatopancreas tissue containing ~14 mg of 1. Overall yields of 1 (52%), 2 (43%), 3 (43%), and 6 (38%) were good, and purities were confirmed by NMR spectroscopy. The structure of 6 was determined by one- and two-dimensional NMR spectroscopy and mass spectrometry. The stability of 6 relative to 1 was also assessed in three solvents in a short-term study that demonstrated the greatest stability in aqueous acetonitrile.     

MM3-ELISA evaluation of coproantigen release and serum antibody production in sheep experimentally infected with Fasciola hepatica and F. gigantica

During an experimental infection of sheep with Fasciola hepatica or F. gigantica, MM3-SERO and MM3-COPRO ELISA tests were applied to compare the kinetics of antibody production and coproantigen release between the 2nd and 32nd week post-infection (wpi). The Kato-Katz technique was used to measure the kinetics of egg shedding by both Fasciola species (eggs per gram of feces, epg). The kinetics of IgG antibodies for all sheep infected with F. hepatica and F. gigantica followed a similar pattern. Optical density (OD) increased rapidly between the 4th until the 12th wpi, when the highest values were reached and then decreased slowly until the 32nd wpi. Coproantigen levels increased above the cut-off value between 6 and 9 wpi in the F. hepatica group, and between 9 and 11 wpi in the F. gigantica group. The comparison between coproantigen levels and epg indicated that F. hepatica-infected sheep had detectable amounts of coproantigens 4–7 weeks before patency (egg shedding), while F. gigantica-infected sheep had detectable amounts of coproantigens 3–6 weeks before patency. When comparing the kinetics of coproantigen release vs the kinetics of epg, a similar pattern emerged, but with a two-week time-lag in epg, for both F. hepatica and F. gigantica infections. The amount of coproantigen release by each adult was not burden dependent for F. hepatica infection (burden of 33–66 adults), while it was for F. gigantica infection (burden of 17–69 adults). The results demonstrate the usefulness of the MM3-SERO and MM3-COPRO ELISAs as tools for the diagnosis of early as well as long-term fascioliasis infections, and suggest that they can potentially be applied to human fascioliasis even in countries where F. hepatica and F. gigantica co-exist. These tests can be employed not only in the diagnosis, but also in studies on epidemiology as well as pathogenesis and treatment in animals and humans since they allow post-treatment infection monitoring.     

Prevalence of pigeon circovirus infections in feral pigeons in Ljubljana, Slovenia

Pigeon circovirus (PiCV) was detected by real-time PCR in cloacal swabs, pharyngeal swabs, and serum samples taken from 74 feral pigeons (Columba livia var. domestica) that were caught at various locations in the city of Ljubljana, Slovenia. PiCV infections were detected in the majority of the tested birds. The highest (74.3%) detection rate was observed in the cloacal swabs and the lowest (31.1%) in serum samples. PiCV DNA was more readily detected in the cloacal swabs, pharyngeal swabs, and serum samples of birds younger than 1 yr. Molecular analysis of partial open reading frame V1 sequences showed that PiCV strains detected in feral pigeons share high nucleotide and amino acid sequence identities with PiCV strains detected in ornamental, racing, meat, and feral pigeons.