Combined application of bacteriophages with a competitive exclusion culture and carvacrol with organic acids can reduce Campylobacter in primary broiler production.

For reducing Campylobacter (C.) in the food production chain and thus the risk to the consumer, the combined application of different measures as a multiple-hurdle approach is currently under discussion. This is the first study to investigate possible synergistic activities in vivo, aiming at reducing intestinal C. jejuni counts by administering (i) bacteriophages (phages) in combination with a competitive exclusion (CE) product and (ii) carvacrol combined with organic acids. The combined application of the two selected phages (Fletchervirus phage NCTC 12673 and Firehammervirus phage vB_CcM-LmqsCPL1/1) and the CE product significantly reduced C. jejuni loads by 1.0 log10 in cecal and colonic contents as well as in cloacal swabs at the end of the trial (33 and 34 days post hatch). The proportion of bacterial isolates showing reduced phage susceptibility ranged from 10.9% (isolates from cecal content) to 47.8% (isolates from cloacal swabs 32 days post hatch) for the Fletchervirus phage, while all tested isolates remained susceptible to the Firehammervirus phage. The use of carvacrol combined with an organic acid blend (sorbic acid, benzoic acid, propionic acid, and acetic acid) significantly reduced Campylobacter counts by 1.0 log10 in cloacal swabs on day 30 only.

Experimental Evidence of Plasmoids in High-ß Magnetic Reconnection.

Magnetic reconnection is a ubiquitous and fundamental process in plasmas by which magnetic fields change their topology and release magnetic energy. Despite decades of research, the physics governing the reconnection process in many parameter regimes remains controversial. Contemporary reconnection theories predict that long, narrow current sheets are susceptible to the tearing instability and split into isolated magnetic islands (or plasmoids), resulting in an enhanced reconnection rate. While several experimental observations of plasmoids in the regime of low-to-intermediate ß (where ß is the ratio of plasma thermal pressure to magnetic pressure) have been made, there is a relative lack of experimental evidence for plasmoids in the high-ß reconnection environments which are typical in many space and astrophysical contexts. Here, we report strong experimental evidence for plasmoid formation in laser-driven high-ß reconnection experiments.

Determining spectral response of the National Ignition Facility particle time of flight diagnostic to x rays.

The Particle Time of Flight (PTOF) diagnostic is a chemical vapor deposition diamond detector used for measuring multiple nuclear bang times at the National Ignition Facility. Due to the non-trivial, polycrystalline structure of these detectors, individual characterization and measurement are required to interrogate the sensitivity and behavior of charge carriers. In this paper, a process is developed for determining the x-ray sensitivity of PTOF detectors and relating it to the intrinsic properties of the detector. We demonstrate that the diamond sample measured has a significant non-homogeneity in its properties, with the charge collection well described by a linear model ax + b, where a = 0.63 ± 0.16 V-1 mm-1 and b = 0.00 ± 0.04 V-1. We also use this method to confirm an electron to hole mobility ratio of 1.5 ± 1.0 and an effective bandgap of 1.8 eV rather than the theoretical 5.5 eV, leading to a large sensitivity increase.

In situ calibration of charged particle spectrometers on the OMEGA Laser Facility using 241Am and 226Ra sources.

Charged particle spectrometry is a critical diagnostic to study inertial-confinement-fusion plasmas and high energy density plasmas. The OMEGA Laser Facility has two fixed magnetic charged particle spectrometers (CPSs) to measure MeV-ions. In situ calibration of these spectrometers was carried out using 241Am and 226Ra alpha emitters. The alpha emission spectrum from the sources was measured independently using surface-barrier detectors (SBDs). The energy dispersion and broadening of the CPS systems were determined by comparing the CPS measured alpha spectrum to that of the SBD. The calibration method significantly constrains the energy dispersion, which was previously obtained through the measurement of charged particle fusion products. Overall, a small shift of 100 keV was observed between previous and the calibration done in this work.

Extension of charged-particle spectrometer capabilities for diagnosing implosions on OMEGA, Z, and the NIF.

New designs and a new analysis technique have been developed for an existing compact charged-particle spectrometer on the NIF and OMEGA. The new analysis technique extends the capabilities of this diagnostic to measure arbitrarily shaped ion spectra down to 1 MeV with yields as low as 106. Three different designs are provided optimized for the measurement of DD protons, T3He deuterons, and 3He3He protons. The designs are highly customizable, and a generalized framework is provided for optimizing the design for alternative applications. Additionally, the understanding of the detector's response and uncertainties is greatly expanded upon. A new calibration procedure is also developed to increase the precision of the measurements.

Characterizing x-ray transmission through filters used in high energy density physics diagnostics.

We report on the design and implementation of a new system used to characterize the energy-dependent x-ray transmission curve, Θ(E), through filters used in high-energy density physics diagnostics. Using an Amptek X-123-CdTe x-ray spectrometer together with a partially depleted silicon surface barrier detector, both the energy spectrum and total emission of an x-ray source have been accurately measured. By coupling these detectors with a custom PROTO-XRD x-ray source with interchangeable cathodes, accurate characterizations of Θ(E) for filters of varying materials and thicknesses have been obtained. The validity of the technique has been confirmed by accurately reproducing areal densities for high-purity filters with known x-ray transmission properties. In this paper, the experimental setup is described and the results of absorption calibrations performed on a variety of different filters are presented.

In vivo efficacy of carvacrol on Campylobacter jejuni prevalence in broiler chickens during an entire fattening period.

Carvacrol, a primary constituent of plant essential oils (EOs), and its antimicrobial activity have been the subject of many in vitro studies. Due to an increasing demand for alternative antimicrobials and an emerging number of antibiotic resistant bacteria, the use of essential oils has played a major role in many recent approaches to reduce Campylobacter colonization in poultry before slaughter age. For that purpose, the reducing effect of carvacrol on Campylobacter jejuni prevalence in broilers was determined in vivo in an experimental broiler chicken model during an entire fattening period. Carvacrol was added to the feed in a concentration of 120 mg/kg feed four days post hatch until the end of the trial. In this study, we demonstrated a statistically significant decrease of C. jejuni counts by 1.17 decadic logarithm (log10) most probable number (MPN)/g in cloacal swabs during starter and grower periods (corresponding to a broilers age between 1 and 28 days). Similar results were observed for colon enumeration at the end of the trial where C. jejuni counts were significantly reduced by 1.25 log10 MPN/g. However, carvacrol did not successfully reduce Campylobacter cecal colonization in 33-day-old broilers.

[Production of monozygotic multiple offspring in agricultural domestic animals]. / Erstellung monozygoter Mehrlinge bei landwirtschaftlichen Nutztieren.

The production of monozygotic twins/multiplets in livestock animal can be achieved either by microsurgical bisection of embryos at the morular- or blastocyst stage, isolation and proliferation of blastomeres from early cleavage stages or nuclear transfer. While the success rates of micro-surgical bisection are high in ruminants (pregnancy rates approximately 50%, twinning rates 20-40%) in polyovulatory species such as swine, the efficiency is low with an average of 20% embryonic survival and 2% monozygotic twins that can positively identified via DNA-fingerprinting. Isolated blastomeres from multicellular embryos still possess great developmental capacity in vitro to progress to the blastocyst stage. However, their development in vivo is markedly reduced. This article summarizes the results obtained by the authors during several years of investigation. The results show for the first time that identical twins can be obtained in pigs which have been demonstrated to be a useful tool in biomedical research.

Ratio of inner cell mass and trophoblastic cells in demi- and intact pig embryos.

Pig morulae, early blastocysts and blastocysts were microsurgically bisected to produce zona-free demi-embryos or remained nonbisected with or without zona pellucida, and the presence of inner cell mass cells was determined using a differential fluorochrome staining technique. After 24 h of in vitro culture, all demi-embryos were classified into three categories, based on morphological criteria: 1, excellent; 2, fair; and 3, degenerated. The average number of total cells and inner cell mass cells in intact embryos cultured without zona pellucida for 24 h was higher (P < 0.05) than that for those with zona pellucida in morulae and early blastocysts. The percentage of demi-embryos without inner cell mass cells in these different morphological categories was 18.7%, 22.2% and 29.8% for morulae, respectively; 3.8%, 16.7% and 30.8% for early blastocysts, respectively; and 3.7%, 32.0% and 36.4% for blastocysts, respectively. The percentage of demi-embryos without inner cell mass cells was lower (P < 0.01) in demi-embryos classified in category 1 compared with category 3 in early blastocysts and in category 1 compared with categories 2 and 3 in blastocysts. Significant differences in the total number of cells and the number of inner cell mass cells were apparent among the three morphological categories of demi-embryos derived from morulae, early blastocysts and blastocysts. The ratio of total cells to inner cell mass cells was similar among intact pig embryos and the different morphological categories of demi-embryos derived from morulae, early blastocysts and blastocysts, with the exception of that between demi-blastocysts of category 1 and the other groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Negatively cis-acting elements in the distal part of the promoter of Epstein-Barr virus trans-activator gene BZLF1.

Epstein-Barr virus (EBV) replicates in a latent or a lytic way in the infected organism, depending on the type and level of differentiation of the host cell. The switch between latency and lytic replication was previously shown, for Burkitt's lymphoma cell lines, to depend on the viral BZLF1 gene product. Protein-DNA assays were used to identify the cis-acting elements that represent the link between regulating signal transduction pathways and the viral cascade of gene expression. Specific binding of proteins to several sites of the BZLF1 promoter during latency was shown. Induction of the lytic cycle by stimulation with 12-O-tetradecanoyl-phorbol 13-acetate abolished the binding of these proteins to the distal promoter (positions -227 to -551), suggesting a functional role for the down-regulation of promoter activity during latency. Computer analysis identified a multiply repeated sequence motif, HI, in this region and exonuclease III footprints confirmed that these sites act as specific protein recognition sites. Using a set of reporter plasmids we were able to demonstrate a negative regulatory effect of the HI motif in some B lymphoid cell lines, in contrast to epithelial HeLa cells. The HI silencer elements are different from other silencer elements described so far in respect of their sequence and protein-binding pattern during the activation of BZLF1.

Generation of identical twin piglets following bisection of embryos at the morula and blastocyst stage.

Porcine morulae and blastocysts were microsurgically bisected and the resulting zona pellucida-free demi-embryos were either cultured in vitro for 48 h or transferred after 24 h of culture into--24 h asynchronous recipients. All demi-embryos were evaluated according to morphological criteria and classified into three categories (excellent, fair or degenerated). The average diameter and the number of cells were determined. Of 1162 bisected embryos, 764 pairs (66%) were evaluated as transferable after 24 h of culture in vitro. The average diameter after 48 h of culture in vitro was different (P < 0.01) among demi-embryos of the three morphological categories as was the number of cells. The greatest diameter and the greatest number of cells were found in demi-embryos classified as morphologically excellent. A total of 22 of 27 recipients (81.5%) remained pregnant and 21 recipients delivered 126 piglets of which six were stillborn. The survival rate of demi-embryos in farrowing recipients was 21.2% (126 of 594). Litter size was significantly reduced in recipients after transfer of demi-embryos compared with that of mated controls (6.0 +/- 2.5 versus 10.8 +/- 2.1 piglets). Similarly, the postpartum losses of piglets were higher in the experimental than in the control gilts (26.7% versus 11.6%). Duration of gestation, average birth weight and daily weight gain were not affected. Among the 126 piglets, seven pairs of identical twins (2.3% of 311 transferred pairs) were identified using several genetic markers in blood (blood groups, polymorphic enzymes and plasma proteins) in a total of 25 gene loci. DNA fingerprinting revealed an identical banding pattern between the two partners of each of the seven pairs. Birth and weaning weight as well as daily weight gain varied considerably between monozygotic partners.

Manipulating early pig embryos.

On the basis of established surgical procedures for embryo recovery and transfer, the early pig embryo can be subjected to various manipulations aimed at a long-term preservation of genetic material, the generation of identical multiplets, the early determination of sex or the alteration of the genetic make-up. Most of these procedures are still at an experimental stage and despite recent considerable progress are far from practical application. Normal piglets have been obtained after cryopreservation of pig blastocysts hatched in vitro, whereas all attempts to freeze embryos with intact zona pellucida have been unsuccessful. Pig embryos at the morula and blastocyst stage can be bisected microsurgically and the resulting demi-embryos possess a high developmental potential in vitro, whereas their development in vivo is impaired. Pregnancy rates are similar (80%) but litter size is reduced compared with intact embryos and twinning rate is approximately 2%. Pig blastomeres isolated from embryos up to the 16-cell stage can be grown in culture and result in normal blastocysts. Normal piglets have been born upon transfer of blastocysts derived from isolated eight-cell blastomeres, clearly underlining the totipotency of this developmental stage. Upon nuclear transfer the developmental capacity of reconstituted pig embryos is low and < 10% develop to morulae or blastocysts in vitro. Pig oocytes can be stimulated parthenogenetically and up to 10% grow to blastocysts in the in vitro culture. Sex determination can be achieved either by separation of X and Y chromosome bearing spermatozoa by flow cytometry or by analysing the expression of the HY antigen in pig embryos from the eight-cell to morula stage. Microinjection of foreign DNA has been successfully used to alter growth and development of transgenic pigs, and to produce foreign proteins in the mammary gland or in the bloodstream, indicating that pigs can be used as donors for valuable human pharmaceutical proteins. Another promising area of gene transfer is the increase of disease resistance in transgenic lines of pigs. Approximately 30% of pig spermatozoa bind considerable amounts of foreign DNA preferably at the post-acrosomal region, suggesting that transgenic animals can be obtained more efficiently than with the usual microinjection procedure. To increase gene transfer efficiency, considerable research efforts have been made to establish embryonic stem (ES) cells, but so far there is no definite proof of totipotency of the generated pig ES-like cells through viable chimaeras. In general, biotechnological procedures are much less advanced in pigs than in cows.

The lytic transition of Epstein-Barr virus is imitated by recombinant B-cells.

Lytic transition of Epstein-Barr virus (EBV) is initiated by distinct immediate early regulators of the viral cycle, in synchronization to temporary, permissive conditions during host cell differentiation. We developed eukaryotic vectors suitable to imitate the processes involved in lytic transition in cell culture systems. Two stable B cell lines were established: R59Z activator cells were used to induce lytic EBV expression in a constitutive manner by the production of the BZLF 1 trans-activator (Zta). R7-57 reporter cells, on the other hand, signaled induced activity of the lytic origin of EBV replication (ori Lyt). Different modes, like chemical induction, lytic superinfection with EBV and single gene trans-activation converted the recombinant ori Lyt element in R7-57 reporter cells. BZLF 1, transiently expressed in R7-57 reporter cells, was the only EBV trans-activator found, sufficient in inducing the viral lytic cycle. Basing on these experiments, trans-cellular activation of EBV was tested by cocultivation of BZLF 1-expressing R59Z activator cells with the R7-57 reporter line. No lytic effect on the reporter cells could be measured, neither by cocultivation of activator cells nor by coincubation of BZLF 1-containing cell lysates. Latency breaking activity, however, was transferred from activator to reporter cells when active, exogenous virus was added. The cell system described in these experiments provides a tool for the detection of EBV reactivation and demonstrates the potential of the lytic regulatory gene BZLF 1.

Waste treatment technologies from the Genesearch Group of companies.

Genesearch is a 10 year old research and development company of Australian scientists, specializing in microbiology and genetics. This research expertise has formed the basis of a number of microbial processes for waste treatment. In addition, a novel type of high yield fermenter for aerobic bacteria allows the economical production of high-potency bacterial preparations for waste treatment processes. A novel approach to the rapid biodegradation of polychlorinated biphenyls has been developed. Photochemical pretreatment partially dechlorinates the molecules, rendering them susceptible to complete and rapid digestion by wild-type soil bacteria. In the area of non-toxic waste, Genesearch has developed products for on-site treatment of, for example, grease-trap wastes and waste oil in ship bilges; and a large scale process for conversion of municipal grease wastes into protein-rich biomass. The prospects for novel biological waste treatment are improving, as public pressure grows, and as increasing government monitoring and penalties make inadequate waste disposal uneconomic.

The nucleotide sequence for the large subunit of ribulose 1,5-bisphosphate carboxylase from a unicellular cyanobacterium, Synechococcus PCC6301.

The gene for the large subunit (LSU) of ribulose 1,5-bisphosphate carboxylase from a unicellular cyanobacterium, Synechococcus PCC6301, was cloned using the spinach LSU gene as a hybridization probe. The coding region of the Synechococcus LSU gene consists of 1419 nucleotides and shows 70% homology to the spinach nucleotide sequence. The derived amino acid sequence (472 amino acids) shows 81% homology to the spinach LSU and 78% to the maize LSU. Regions containing active-site residues are highly conserved among spinach, maize, and Synechococcus. In contrast, the first 13 amino acids are poorly conserved (30% homology), supporting the hypothesis that this region is proteolytically removed. The 5'-flanking region of the Synechococcus LSU gene contains sequences which correspond to bacterial consensus sequences for the -35 region and Pribnow box. Two 11-bp sequences in the 5' region show high homology to sequences in spinach and maize. One of these encompasses a possible ribosome-binding site. The 3'-flanking region contains a 35-bp sequence capable of giving rise to a terminator structure.

Isolation, properties, function, and regulation of endo-(1 leads to 3)-beta-glucanases in Schizosaccharomyces pombe.

Cell-free extracts, membranous fractions, and cell wall preparations from Schizosaccharomyces pombe were examined for the presence of (1 --> 3)-beta-, (1 --> 3)-alpha-, and (1 --> 6)-beta-glucanase activities. The various glucanases were assayed in cells at different growth stages. Only (1 --> 3)-beta-glucanase activity was found, and this was associated with the cell wall fraction. Chromatographic fractionation of the crude enzyme revealed two endo-(1 --> 3)-beta-glucanases, designated as glucanase I and glucanase II. Glucanase I consisted of two subunits of molecular weights 78,500 and 82,000, and glucanase II was a single polypeptide of 75,000. Although both enzymes had similar substrate specificities and similar hydrolytic action on laminarin, glucanase II had much higher hydrolytic activity on isolated cell walls of S. pombe. On the basis of differential lytic activity on cell walls, glucanase II was shown to be present in conjugating cells and highest in sporulating cells. Glucanase II appeared to be specifically involved in conjugation and sporulation since vegetative cells and nonconjugating and nonsporulating cells did not contain this enzyme. The appearance of glucanase II in conjugating cells may be due to de novo enzyme synthesis since no activation could be demonstrated by combining extracts from vegetative and conjugating cells. Increased glucanase activity occurred when walls from conjugating cells were combined with walls from sporulating cells. Studies with trypsin and proteolytic inhibitors suggest that glucanase II exists as a zymogen in conjugating cells. A temperature-sensitive mutant of S. pombe was isolated which lysed at 37 degrees C. Glucanase activity was higher in vegetative cells held at 37 degrees C than cells held at 25 degrees C. Unlike the wild-type strain, this mutant contained glucanase II activity during vegetative growth and may be a regulatory mutant.