Identification of transgenes from wood of genetically transformed poplar trees

We have extracted total DNA from different fractions of fresh wood as well as from cold-stored and air-dried wood harvested from transgenic aspen grown in the field. The highest amounts of DNA were obtained from bark/cambium tissue; the DNA quality, however, was poor. Best results in PCR and Southern blot analyses were obtained from DNA extracted from early wood. Using appropriate primer pairs, amplification products were obtained from both the foreign gene (transgene) and aspen genomic sequences. In Southern blot analyses transgene-specific hybridisation signals were obtained. This is the first report on the detection of foreign genes in wood sampled from genetically modified trees.     

Tissue tropism of Helicobacter pullorum in specific pathogen-free chickens determined by culture and nucleic acid detection

Three-day-old specific pathogen-free chickens (n = 24) located in isolators were inoculated orally with Helicobacter pullorum. One group (n = 12) was infected with a H. pullorum field isolate from human origin, another one (n = 12) with the American Type Culture Collection H. pullorum reference isolate 51801 originating from chickens. Both isolates were positive for cytolethal distending toxin, investigated using a polymerase chain reaction (PCR). A third group (n = 4) was kept as a negative control. Starting on day 7 of life, birds from each group were euthanatized at different time points up to 35 days. Various organ samples were taken aseptically and processed by culture and a H. pullorum-specific PCR. In the group infected with the human isolate the nucleic acid of H. pullorum was detected in the caecal tonsils and caeca of 12 and 11 birds, respectively. Live bacteria were cultivated from the caecal tonsils and caeca of five birds 24 and 31 days postinfection. Live bacteria were also isolated from the heart of one bird, whereas PCR had to be used to detect the nucleic acid of H. pullorum in the gallbladder of four birds. No live bacteria were reisolated at any time from birds infected with the avian isolate, but bacterial nucleic acid was detected in the caeca of five birds and in the gallbladder of one. In both groups neither live H. pullorum nor its nucleic acid were detected in the liver, spleen, and duodenum. Compared to the avian H. pullorum isolate the human isolate proved to be more invasive. No obvious clinical symptoms or disease was seen in the chickens during the entire experiment. The reisolation of live bacteria at the end of the experiments indicates that H. pullorum could enter the food chain even after early infection in birds. Furthermore, PCR was demonstrated to be helpful in tracing these fastidious bacteria.     

Prevalence of Campylobacter spp. in poultry meat and raw milk using PCR, conventional cultural methods and Fourier transform infrared spectroscopy

The identification of thermotolerant campylobacters in official food control in the state of Baden-Wuerttemberg has been traditionally performed using the cultural procedure as described in the ISO-Norm 10272:1995. Analysis thus took 5-6 days to complete. Additionally diagnostic problems caused by the accompanying flora as well as the resistance to nalidixic acid occured. Within the scope of this study these problems could be solved by introducing a filtration step for the reduction of the accompanying flora and by performing the indoxyl acetate-hydrolysis-test in addition to the antibiotic-resistance-test. Besides various PCR protocols for the identification of thermotolerant campylobacters from food were established as an alternative to the cultural procedure, providing reliable results within two days. Furthermore, infrared spectroscopy was tested for the identification of Campylobacter isolates. Using this technique and with the help of a suitable data base, bacterial pure cultures can be differentiated within 2 hours. Among others 356 samples of raw poultry meat were tested with the newly established procedures as well as with the classical cultural method, showing that 32% of the samples were Campylobacter spp. positive. 37% of these isolates were resistant against nalidixic acid. This indicates that the development of resistances in Campylobacter spp. in Germany follows the same trend described for other European countries.     

Site and extent of digestion, duodenal flow, and intestinal disappearance of total and esterified fatty acids in sheep fed a high-concentrate diet supplemented with high-linoleate safflower oil

Our objective was to determine duodenal and ileal flows of total and esterified fatty acids and to determine ruminal fermentation characteristics and site and extent of nutrient digestion in sheep fed an 80% concentrate diet supplemented with high-linoleate (77%) safflower oil at 0, 3, 6, and 9% of DM. Oil was infused intraruminally along with an isonitrogenous basal diet (fed at 2% of BW) that contained bromegrass hay, cracked corn, corn gluten meal, urea, and limestone. Four crossbred wethers (BW = 44.3 +/- 15.7 kg) fitted with ruminal, duodenal, and ileal cannulas were used in a 4 x 4 Latin square experiment, in which 14 d of dietary adaptation were followed by 4 d of duodenal, ileal, and ruminal sampling. Fatty acid intake increased (linear, P = 0.004 to 0.001) with increased dietary safflower oil. Digestibilities of OM, NDF, and N were not affected (P = 0.09 to 0.65) by increased dietary safflower oil. For total fatty acids (free plus esterified) and esterified fatty acids, duodenal flow of most fatty acids, including 18:2c-9,c-12, increased (P = 0.006 to 0.05) with increased dietary oil. Within each treatment, duodenal flow of total and esterified 18:2c-9,c-12 was similar (P = 0.32), indicating that duodenal flow of this fatty acid occurred because most of it remained esterified. Duodenal flow of esterified 18:1t-11 increased (P = 0.08) with increased dietary safflower oil, indicating that reesterification of ruminal fatty acids occurred. Apparent small intestinal disappearance of most fatty acids was not affected (P = 0.19 to 0.98) by increased dietary safflower oil, but increased (P = 0.05) for 18:2c-9,c-12, which ranged from 87.0 to 97.4%, and for 18:2c-9,t-11 (P = 0.03), which ranged from 37.9% with no added oil to 99.2% with supplemental oil. For esterified fatty acids, apparent small intestinal disappearance was from 80% for 18:3c-9,c-12,c-15 at the greatest level of dietary oil up to 100% for 18:1t-11 and 18:1c-12 with 0% oil. We concluded that duodenal flow of 18:2c-9,c-12 was predominately associated with the esterified fraction, suggesting that the extent of ruminal lipolysis was decreased with increased dietary high-linoleate safflower oil. Furthermore, biohydrogenation intermediates observed in the esterified fatty acids indicated that some reesterification occurred, and the high level of apparent absorption of esterified fatty acids indicated that intestinal lipolysis did not limit overall digestion of the fatty acids fed to the sheep.     

Mechanism of wine lactone formation: demonstration of stereoselective cyclization and 1,3-hydride shift

The cyclization mechanism of (E)-2,6-dimethyl-6-hydroxyocta-2,7-dienoic acid to wine lactone under acidic aqueous conditions was investigated using the two stereoselectively deuterium-labeled precursors (2E,6R,7Z)-[8-2H]-2,6-dimethyl-6-hydroxyocta-2,7-dienoic acid and (2E,7E)-(+/-)-[8-2H]-2,6-dimethyl-6-hydroxyocta-2,7-dienoic acid. A detailed analysis of the generated wine lactone isomers by enantioselective multidimensional gas chromatography (MDGC)/ion trap tandem mass spectrometry demonstrates that the formation of wine lactone proceeds via a nonenzymatic stereoselective cationic cyclization cascade that includes a 1,3-hydride shift. Usually, such mechanisms are features of cyclization reactions that are catalyzed by terpene cyclases. This nonenzymatic conversion of an acyclic precursor to a bicyclic monoterpene under relevant cationic cyclization conditions has rarely been observed and confirms recent suggestions that the precursor itself can provide the chemical functionality required for specific steps in the cyclization cascade.     

Virulence genotype of Pasteurella multocida strains isolated from different hosts with various disease status

To learn more about the molecular biology of Pasteurella multocida 289 strains isolated from various clinically healthy and diseased hosts were examined for capsule biosynthesis genes (capA, B, D, E, and F) and 14 virulence associated genes by PCR and DNA-DNA-hybridization. As expected, capsule type A strains were highly adapted to bovines (92.3%) and poultry (85.7%) while we mainly found capA (34.9%)- and capD (58.1%)-positive strains in swine. A noticeable amount of capD-positive strains also originated from small ruminants (34.9%) and capF was detected in wild type strains from diseased cattle (2.2%) and cats (7.4%). None of the isolates harboured capE, while capB was exclusively found in all strains from buffaloes. Nearly all isolates showed a combination of genes encoding outer membrane proteins, colonization factors, iron aquisition factors and superoxid-dismutases without any clue for host specificity. In contrast, the transferrin binding protein encoding gene tbpA (31.5%) was limited to ruminant strains and only 37.0% of all P. multocida strains harboured pfhA, coding for a filamentous hemagglutinin, supposed to be a putative adhesion- und serum resistance factor. PfhA revealed a strong positive association to the outcome of disease in bovine hosts and in combination with toxA to that in swine. The dermonecrotoxin encoding toxA, present in 12.5% of all strains, was detected in isolates from swine, small ruminants, cattle, and poultry. A significant association to the disease status, however, was only existent in swine, although with 66.7% we found a notably high prevalence of the toxin gene among strains from small ruminants. The genes toxA, tbpA and pfhA as well as capsule biosynthesis genes are supposed to be important epidemiological marker genes for characterizing P. multocida field strains.     

Simple prediction of the survival of follicles in cryopreserved human ovarian tissue

This study examines the possible predictive value of the LIVE/DEAD fluorescence viability assay for evaluation of survival of cryopreserved human ovarian tissue. Ovarian tissue from ten patients was examined by LIVE/DEAD viability staining before and after cryopreservation and after freezing in a -20 C freezer (negative control). After cryopreservation with a slow freezing protocol and cryoprotectant the LIVE/DEAD assay showed 86% viable follicles (an intact oocyte and at least more than 50% of the granulosa cells alive), whereas after freezing at -20 C the survival rate was 67%. The healthy follicular loss after cryopreservation was 4%, whereas with freezing at -20 C, it was 25%. Although this assay overestimates the survival rate of cryopreserved primordial follicles, if the LIVE/DEAD assay yields greater than approximately 85% viable follicles, it can be assumed that the follicles in the cryopreserved tissue have maintained their developmental potential and that the tissue is suitable for retransplantation.     

Detection and identification of food-borne pathogens of the genera Campylobacter, Arcobacter and Helicobacter by multiplex PCR in poultry and poultry products

The objective of this study was to develop a multiplex polymerase chain reaction (PCR) to detect and differentiate food-borne pathogens of the three genera Campylobacter, Arcobacter and Helicobacter in a single step procedure. One common reverse primer and three genus-specific forward primers were designed by hybridizing to the 16S rRNA of selected reference strains. Besides the species with significance as food-borne pathogens isolated from poultry meat--Campylobacter jejuni, Campylobacter coli, Arcobacter butzleri and Helicobacter pullorum--several other members of these genera were tested to determine the specificity of the designed multiplex PCR. In total, 20 ATCC and NCTC reference strains of Campyobacter, Arcobacter and Helicobacter were used to evaluate the PCR. Specific amplificates were obtained from all thermophilic species of Campylobacter as well as from species of Arcobacter and Helicobacter. No amplification product was obtained from the non-thermophilic Campylobacter, C. hyointestinalis and C. fetus. Furthermore, a total of 43 field strains of the three genera isolated from poultry, pigs, cattle and humans were investigated using this PCR. To confirm the classification of 10 H. pullorum strains the 16S rRNAs were sequenced. The developed PCR is a helpful diagnostic tool to detect and differentiate Campylobacter, Arcobacter and Helicobacter isolated from poultry and poultry products.