Enzymatic production and complete nuclear magnetic resonance assignment of the sugar lactulose

The enzymatic transgalactosylation from lactose to fructose leading to the prebiotic disaccharide lactulose was investigated using the beta-galactosidase from Aspergillus oryzae and the hyperthermostable beta-glycosidase from Pyrococcus furiosus (CelB). The conditions for highest lactulose yields relative to the initial lactose concentration were established on a 1 mL scale. Dependent on the initial molar ratio of lactose to fructose, more or fewer oligosaccharides other than lactulose were generated. Bioconversions on a 30 mL scale in a stirred glass reactor were performed, and lactulose yields of 46 mmol/L (44% relative to lactose) for CelB and 30 mmol/L (30% relative to lactose) for A. oryzae beta-galactosidase were achieved. Only <5% of other oligosaccharides were detectable. The corresponding productivities were 24 and 16 mmol/L/h, respectively. The molecular structure of lactulose was investigated in detail and confirmed after purification of the reaction solution by LC-MS and 1D and 2D NMR. Lactulose (4-O-beta-D-galactopyranosyl-D-fructose) was unambiguously proved to be the major transglycosylation disaccharide.     

Detection of non‐equilibrium contaminant release in soil columns: Delineation of experimental conditions by numerical simulations

Robust and reliable laboratory methods are required to assess the rate of release of pollutants from contaminated materials. A widely used approach to determine release parameters are soil column experiments. Release rates are thereby obtained by the analysis of the concentration time profile measured at the column outlet, the so called breakthrough curve. Insufficient experimental information as a consequence of simple experimental designs, however, results in the non‐unique identification of the release processes. This is due to the equifinality, i.e., different processes result in the same breakthrough behavior. We analyzed column outflow experiments for their suitability for the identification of non‐equilibrium conditions by means of numerical simulations. Results show that non‐equilibrium conditions can be detected only in a small range of release time scale to transport time scale. Most frequently, non‐equilibrium remains undetected due to unmatched experimental conditions. Flow interruptions and modulation of the flow velocity are an important means to allow for better detection of possible non‐equilibrium release processes. The efficiency of flow interruptions sensitively depends on the experimental conditions, such as point of time and duration of the interruption and the imposed flow velocity. Column experiments are a suitable experimental tool to investigate non‐equilibrium. However, the course of the experiment has to be designed very carefully.     

Bilateral antler sequestration above the coronet in a red deer (Cervus elaphus) stag—Insights into the process of antler casting

This paper reports a case of delayed velvet shedding and bilateral premature antler casting above the coronets in a young adult red deer stag from Germany. Based on the established role of testosterone in the control of the antler cycle, the antler abnormality is considered to have been the result of a (temporary) androgen deficiency. The basal surfaces (separation planes or seals) of the cast antlers were markedly concave. Scanning electron microscopy revealed that the separation plane was densely covered with Howship's lacunae, denoting intense osteoclastic activity along the border between the proximal (living) and distal (dead) antler portions. Our observations and those of previous studies indicate that antler casting does not occur at a pre-determined separation plane, but along the border between living and dead bone, regardless of the position of this border within the cranial appendages. This is a major difference to autotomy of (living) appendages at fixed breakage planes, as it occurs for instance in lizard tails.     

Comparing validation of four ELISA-systems for detection of Salmonella derby- and Salmonella infantis-infected pigs

The objective of this study was the comparative evaluation of four indirect Salmonella ELISA tests at study time approved in Germany to detect Salmonella infection in pigs.Three tests are based on a LPS-antigen mix and directed against specific IgG antibodies. The fourth test is based on a purified S. Typhimurium whole-cell lysate antigen and discriminates between Salmonella-specific IgM-, IgA-, and IgG- antibodies. In a longitudinal study, two groups of six weeks old hybrid piglets were orally infected with a porcine S. Infantis or S. Derby strain. Clinical and bacteriological parameters were monitored weekly during an observation period of 130 days after infection and serum samples were investigated in parallel with the respective ELISAs. Apparently, the LPS-based ELISA systems used in this study failed to recognize S. Infantis-infected pigs although those animals shed the pathogen in high amounts throughout the study until day 81 post infection (p. i.). In contrast, the isotype-specific Salmonella Typhimurium whole-cell-lysate based ELISA was capable of detecting Salmonella-infected pigs from day ten p. i. at all tested serotypes and revealed the highest sensitivity in detection of S. Infantis-infected pigs. Furthermore, it became apparent that the often used surveillance cut-off value of 40 OD% is not appropriate for intra-vitam detection of S. Infantis- and S. Derby-infected pigs. In contrast, the cut-off values of the ELISAs given by the suppliers result in considerable higher detection rates.     

Two outbreaks of virulent systemic feline calicivirus infection in cats in Germany

Over the last years, several outbreaks of virulent systemic feline calicivirus (VS-FCV) infection have been described in the USA and several European countries. The paper describes two outbreaks of VS-FCV infection in cats in Germany. Data concerning clinical, laboratory, and histopathological features ofVS-FCV infection were collected from two outbreaks affecting 55 and 4 cats, respectively. Presence of feline calicivirus was confirmed by PCR followed by sequencing of the PCR-products. Clinical signs were variable, including severe upper respiratory tract infection, dyspnoea, oral and footpad ulceration, facial oedema, enteritis, pneumonia, bleeding disorder, high fever, and icterus. Both outbreaks were characterized by a high mortality rate.The present report describes the first documented outbreaks of VS-FCV infection in cats in Germany. Clinical and histopathological features are comparable to outbreaks described in the USA and Europe. However, phylogenetic analysis of the virus genome suggests that virus strains involved in these outbreaks were different from each other and from virulent strains isolated before, confirming the known genetic variability of FCV.     

First isolation and characterisation of Encephalitozoon cuniculi from a free-ranging rat (Rattus norvegicus)

The microsporidian species Encephalitozoon cuniculi can infect a wide variety of mammals including man. It is a common parasite in rabbits and several sporadic infections in laboratory rats have been described. Based on molecular data three E. cuniculi strains have been identified. Here we describe the first in vitro propagation of E. cuniculi, which was isolated from a free-ranging rat (Rattus norvegicus). The rat was one of three seropositive animals among 23 rats captured in the city of Zurich. The new isolate was further characterised as strain II ("mouse"-strain) based on the rDNA internal transcribed spacer sequence. Western blot analysis of this isolate revealed slight differences to other available strain II isolates originating from laboratory mice and farmed blue foxes. The new isolate caused disseminated infection in liver and lung upon oral inoculation of Brown Norway (BN) rats and was transmitted to sentinel rats. This rat-adapted isolate will be valuable to study the pathogenesis of Encephalitozoon infections in the rat model.     

Validierung der Extraktion von Nematodenzysten aus Bodenproben mit dem MEKU-Bodenextraktor

The diagnosis of phytopathogenic agents has to be justiciable. Quality management gains increasing importance. In preparation of accreditation of the diagnostic laboratory at the Agricultural Public Service Center Rheinhessen-Nahe-Hunsrück in Bad Kreuznach the extraction of cyst nematodes from soil samples by means of a MEKU-soil extractor was validated according to the principles of the German Accreditation Association (DAkkS). The results achieved with test procedure were analyzed according to the parameters analytical sensitivity, analytical specification, selectivity, repeatability and reproducibility. The limit of acceptance kept the conditions of the quality control plan. Therefore the test procedure is qualified for diagnosis of quarantine organisms according to the specifications of DAkkS. Continuous participation in laboratory comparative tests ensures a long term, high level quality standard.