Release of C and N from roots of peas and oats and their availability to soil microorganisms

Nutrient mobilisation in the rhizosphere is driven by soil microorganisms and controlled by the release of available C compounds from roots. It is not known how the quality of release influences this process in situ. Therefore, the present study was conducted to investigate the amount and turnover of rhizodeposition, in this study defined as root-derived C or N present in the soil after removal of roots and root fragments, released at different growth stages of peas (Pisum sativum L.) and oats (Avena sativa L.). Plants were grown in soil columns placed in a raised bed under outdoor conditions and simultaneously pulse labelled in situ with a ¹³C-glucose-¹⁵N-urea solution using a stem feeding method. After harvest, ¹³C and ¹⁵N was recovered in plant parts and soil pools, including the microbial biomass. Net rhizodeposition of C and N as a percentage of total plant C and N was higher in peas than in oats. Moreover, the C-to-N ratio of the rhizodeposits was lower in peas, and a higher proportion of the microbial biomass and inorganic N was derived from rhizodeposition. These results suggest a positive plant-soil feedback shaping nutrient mobilisation. This process is driven by the C and N supply of roots, which has a higher availability in peas than in oats.     

Identification and immunological characterization of conserved Mycoplasma hyopneumoniae lipoproteins Mhp378 and Mhp651

Mycoplasma hyopneumoniae, the etiological agent of swine enzootic pneumonia, is an important pathogen in the swine industry worldwide. Investigations on pathogenicity mechanisms as well as current serological detection methods and the development of new recombinant subunit vaccines are hampered by the lack of known and well characterized, species-specific M. hyopneumoniae antigens. As a first step to solve these problems membrane and membrane-associated proteins were enriched from M. hyopneumoniae cells by Triton X-114 fractionation and further analyzed by 2D gel electrophoresis and Western blot analyses using convalescent sera. Two previously unknown immunogenic proteins were identified by quadrupole time-of-flight mass spectrometry and database analyses as the conserved putative lipoproteins, Mhp378 and Mhp651. Both proteins were expressed as recombinant GST fusion proteins and reacted with sera from convalescent pigs. Coated as solid-phase antigen, Mhp651 showed a distinct cross-reaction only with Mycoplasma flocculare specific rabbit hyperimmune serum, whereas Mhp378 was only recognized by the positive control serum directed against M. hyopneumoniae, thereby indicating its species specificity.     

Sonication-assisted extraction of chitin from North Atlantic shrimps (Pandalus borealis)

The influence of sonication during extraction of chitin from North Atlantic shrimp (NAS) shells (Pandalus borealis) on chitin yield, purity, and crystallinity was investigated. Shells were peeled, washed, lyophilized, ground, and suspended for 4 h in 0.25 M HCl (1:40) at 40 degrees C followed by ultrasonication at 41 W/cm(2) for 0, 1, and 4 h, respectively. Demineralized shells were lyophilized, resuspended in 0.25 M NaOH (1:40), and ultrasonicated at 41 W/cm(2) for 0, 1, and 4 h to remove proteins. The yield and mineral and protein contents were determined after each processing step. The purity of extracted chitin was determined from the total amount of glucosamine. The crystallinity index and size of crystals were calculated from wide-angle X-ray scattering measurements. Scanning electron microscope images were recorded to evaluate morphological changes in samples. The yield of chitin from NAS decreased from 16.5 to 11.4% for 0 and 1 h sonicated samples, respectively, which was attributed to increased concentrations of depolymerized materials in the wash water. Sonication did not enhance the removal of minerals. The application of ultrasound enhanced the removal of proteins from 39.8 to 10.6, 8.3, and 7.3% after 0, 1, and 4 h of sonication treatments. The crystallinity index of chitin decreased from 87.6 to 79.1 and 78.5% after 1 and 4 h of sonication, yielding chitosans with crystallinity indices of 76.7, 79.5, and 74.8% after deacetylation, respectively. Fourier transform infrared spectroscopy scans indicated that the degree of acetylation of chitins was unaffected by sonication. Comparison of the extraction results of NAS with that from freshwater prawns indicated that more impurities were left in NAS chitin, suggesting that composition and structural arrangement of chitin in shells influence the efficiency of ultrasound-assisted extraction.     

Sonication-assisted extraction of chitin from shells of fresh water prawns (Macrobrachium rosenbergii)

The effect of sonication during chitin extraction from freshwater prawn shells on yield, purity, and crystallinity of chitin was investigated. Dry prawn shells were suspended for 4 h in 0.25 M HCl at 40 degrees C while they were sonicated for 0, 1, and 4 h. Demineralized shells were lyophilized, resuspended in 0.25 M NaOH, and sonicated again for 0, 1, and 4 h for protein removal. The yield of chitin decreased from 8.28 to 5.02% for nonsonicated and sonicated samples, respectively, which was attributed to losses of depolymerized materials in the wash water. The application of ultrasound enhanced the removal of proteins. In nontreated shells, the amount of protein was 44.01% and was reduced to 12.55, 10.59, and 7.45% after 0, 1, and 4 h of sonication treatments. The glucosamine content slightly decreased with sonication probably because of losses due to depolymerization. The crystallinity indices of chitins decreased as the time of sonication increased. The degree of acetylation of chitins was unaffected by sonication, but the degree of acetylation of chitosans produced from sonicated chitin decreased from 70.0 to 68.7 and 61.4% for 1 and 4 h sonicated samples, respectively.     

Detection of avian rotaviruses of groups A, D, F and G in diseased chickens and turkeys from Europe and Bangladesh

Avian rotaviruses (AvRVs) represent a diverse group of intestinal viruses, which are suspected as the cause of several diseases in poultry with symptoms of diarrhoea, growth retardation or runting and stunting syndrome (RSS). To assess the distribution of AvRVs in chickens and turkeys, we have developed specific PCR protocols. These protocols were applied in two field studies investigating faecal samples or intestinal contents of diseased birds derived from several European countries and Bangladesh. In the first study, samples of 166 chickens and 33 turkeys collected between 2005 and 2008 were tested by PAGE and conventional RT-PCR and AvRVs were detected in 46.2%. In detail, 16.1% and 39.2% were positive for AvRVs of groups A or D, respectively. 11.1% of the samples contained both of them and only four samples (2.0%) contained rotaviruses showing a PAGE pattern typical for groups F and G. In the second study, samples from 375 chickens and 18 turkeys collected between 2009 and 2010 were analyzed using a more sensitive group A-specific and a new group D-specific real-time RT-PCR. In this survey, 85.0% were AvRV-positive, 58.8% for group A AvRVs, 65.9% for group D AvRVs and 38.9% for both of them. Although geographical differences exist, the results generally indicate a very high prevalence of group A and D rotaviruses in chicken and turkey flocks with cases of diarrhoea, growth retardation or RSS. The newly developed diagnostic tools will help to investigate the epidemiology and clinical significance of AvRV infections in poultry.     

Landscape occupancy and local population size depends on host plant distribution in the butterfly Cupido minimus

We tested whether the landscape occupancy and local population size of the monophagous butterfly Cupido minimus can be predicted by patch size and isolation of its host plant or by other habitat characteristics. C. minimus and its larval food plant Anthyllis vulneraria are classified as rare and endangered in northern Germany. Adults of C. minimus are ranked as the most sedentary butterfly species in northern Europe.Around the city of Göttingen (Germany), we checked all known locations of A. vulneraria (n=70) in June 2002 for butterfly eggs (in blooming flowerheads) and adult butterflies (within 20-min transects).We found eggs of C. minimus or a high number of adults (>7) in all habitats with A. vulneraria (which are calcareous grasslands) even when isolated up to 2-4 km. In multiple regression analyses, local population size of adult butterflies was positively related to the cover of its larval food plant A. vulneraria explaining 65% of variance. Cover of A. vulneraria increased with increasing habitat area and increasing cover of plant species in flower and decreased with increasing cover of shrub layer. Habitat isolation and further factors describing habitat quality were not related to C. minimus population size or cover of its larval food plant.The results suggest that dispersal ability of C. minimus is greater than expected and that management should focus to increase A. vulneraria patches. For conservation, low impact grazing once a year and removing of excessive shrubs in winter seems to be the most appropriate strategies.     

Evaluation of serological tests for detecting tick-borne encephalitis virus (TBEV) antibodies in animals

Tick-borne encephalitis (TBE) in animals is not well understood yet. TBE virus (TBEV) serology in several host species could be valuable for epidemiological analyses in the field as well as for the detection of clinical cases. However, performance and suitability of the available test systems are not well assessed. Therefore, we evaluated two commercial TBEV-ELISA kits in a pilot study and compared them for their suitability in veterinary applications. For this purpose, we tested 163 field collected goat sera and evaluated the results by serum neutralization test (SNT) as "gold standard". Twenty-eight SNT positive sera (17.2%) were detected. The best suited ELISA kit was used for determination of a species-specific cutoff for horses, cattle, sheep, goats, pigs, mice, dogs, rabbits and monkeys with defined sera from animals without known or with improbable contact to TBEV. The level of non-specific ELISA results does not only differ between animal species but may also be influenced by the age of the tested animals. The number of sera which tested false positive by ELISA was higher in older than in young sheep. In order to obtain defined polyclonal sera as references, two dogs, cattle, goats, sheep, rabbits and pigs each, as well as one horse and 90 mice were immunized four times with a commercially available TBEV vaccine. In conclusion, our results demonstrated that commercial TBEV-ELISA kits are suitable for application in veterinary medicine for both, verification of clinical TBE cases and epidemiological screening. However, positive ELISA results should be verified by SNT. Only a very low number of false negative ELISA-results were found.     

Isolation of scFv fragments specific to OmpD of Salmonella Typhimurium

Pork meat is one of the major sources for human infections with Salmonella enterica subspecies enterica serovars. Further, zoonoses caused by S. enterica subspecies enterica serovars are responsible for substantial economical losses in industrial countries. Quick and reliable detection of this infection is urgently needed to improve consumer security. Due to its capability to identify infections independent of the species, a competitive ELISA is the preferable method for the detection of anti-Salmonella antibodies in serum. Recombinant antibody fragments (scFvs) were isolated from the naive human antibody gene library HAL7 by phage display. Recombinant produced outer membrane protein D (OmpD) of Salmonella Typhimurium was used as antigen. The characterization of the isolated single chain Fv (scFv) antibodies was done by enzyme-linked immunosorbent assay (ELISA), immunoblot, sequencing, epitope mapping and size exclusion chromatography (SEC). The detection of anti-OmpD IgGs in swine sera by competitive ELISA was shown in a proof of principle concept. Furthermore, the developed competitive ELISA would be compatible to a recently published DIVA vaccine, allow to distinguish between infected and vaccinated pigs.     

Investigation of the volatile composition of pinotage wines fermented with different malolactic starter cultures using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOF-MS)

Headspace solid phase microextraction in combination with comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (HS-SPME-GC × GC-TOF-MS) was used for the detailed investigation of the impact of malolactic fermentation (MLF) using three commercial Oenococcus oeni strains on the volatile composition of Vitis vinifera cv. Pinotage wines. GC × GC allowed the identification of 115 volatile compounds, including both major constituents and trace-level compounds, in a single analysis. A number of compounds differing in mean concentration levels between the control wines and those fermented with different starter cultures were shown for the first time to be influenced by MLF and/or the bacterial strain. Principal component analysis (PCA) provided excellent separation between the wines fermented with different MLF starter cultures and the control wine. Significantly different levels for some volatile compounds in wines fermented with one of the LAB starter cultures could be indicative of metabolic differences of this strain.