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    ABSTRACT: A cellular logic system capable of combinatorial and sequential logic operations based on bacterial protein-triggered cytotoxicity was constructed. Advanced devices such as a keypad lock, half-adder and several basic Boolean properties were demonstrated on the cells.
    Chemical Communications 11/2013;
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    ABSTRACT: A comparative analysis on the relevance of the Bacillus cereus enterotoxins Nhe (nonhemolytic enterotoxin), HBL (hemolysin BL) and CytK (cytotoxin K) was accomplished, concerning their toxic activity towards different target cell lines. Overall, among the components secreted by the reference strains for Nhe and HBL, the enterotoxin complexes accounted for over 90 % of the total toxicity. Vero and primary endothelial cells (HUVEC) were highly susceptible to Nhe, whereas Hep-G2, Vero and A549 reacted most sensitive to Nhe plus HBL. For CytK the highest toxicity was observed on CaCo-2 cells. As HBL positive strains always produce Nhe in parallel, the specific contribution of both enterotoxin complexes to the overall observed cytotoxic effects was determined by consecutively removing their single components. While in most cell lines Nhe and HBL contributed more or less equally (40-60 %) to cytotoxicity, the relative activity of Nhe was approximately 90 % in HUVEC, and that of HBL 75 % in A549 cells. With U937, a nearly Nhe resistant cell line was identified for the first time. This distinct susceptibility of cell lines was confirmed by investigating a set of 37 B. cereus strains. Interestingly, whereas Nhe is the enterotoxin mainly responsible for cell death as determined by WST-1 bioassays, more rapid pore formation was observed when HBL was present, pointing to a different mode of action of the two enterotoxin complexes. Furthermore, correlation was observed between cytotoxicity of solely Nhe producing strains and NheB. Cytotoxicity of Nhe/HBL producing isolates correlated with the expression of HBL L1, NheB and HBL B. In conclusion, the observed susceptibilities of target cell lines of different histological origin underline that B. cereus enterotoxins represent major virulence factors and that toxicity is not restricted to gastrointestinal infections. The varying contribution of Nhe and HBL to total cytotoxicity strongly indicates that Nhe as well as HBL specific B. cereus enterotoxin receptors exist.
    Toxicon 11/2013;
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    ABSTRACT: The welfare of an animal is ensured if it is able to fully satisfy its essential species-typical needs in all functional aspects of behaviour. In mink, stereotypies and apathy, internal and/or external injuries as well as increased susceptibility to disease have been known to occur as a result of chronic stress. The non-invasive method of analysing faecal cortisol metabolites (FCM) allows conclusions to be drawn about the stress level in the respective housing system. The objective of this study is to find out how the cortisol metabolites content in the faecal changes with increasing age of the mink under semi-natural housing conditions. Thus, 40 American mink (Neovison vison) were housed in two outdoor enclosures imitating natural conditions. Throughout the entire study (13th to 32nd week of life), faecal samples were collected to measure cortisol metabolites. No differences in FCM concentrations between the two outdoor enclosures were found. In the young mink lower, less fluctuating FCM levels were found than in older animals. After the first faecal collection in the 13th/14th week of life, the level of metabolites decreased slightly (p = 0.032; 17th/18th week). From the 22nd/23rd week onwards until the 30th/31st week, shortly before the animals were pelted, continuously increasing concentrations were then measured. Increasing FCM levels with advancing age of the animals are probably attributable to the onset of sexual maturity and/or the respective season. This has to be taken into account in future studies using this method for assessing welfare and when comparing different mink housing systems.
    J Anim Physiol a Anim Nutr 09/2013; 98:497-503.
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    ABSTRACT: We present an OR gate based on monoclonal antibodies for the simultaneous detection of multiple toxins in a single tube. To further simplify the operating procedure, the Boolean rule of simplification was used to guide the selection of a marker toxin among the natural toxin profiles.
    Chemical Communications 09/2013;
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    ABSTRACT: In the previously performed field study from 2007 to 2009, it became evident that foot pad alterations were already commonly found in turkeys at the age of 6 wk. At this early age, 45% of the clinically examined birds were diagnosed with epithelial necrosis. Therefore, it became important to specifically analyze the situation during the early rearing phase. The present study reflects the prevalence and severity of foot pad alterations of turkey poults up to the age of 35 d (5 wk), starting as early as the age of 3 d. From 24 turkey farms throughout Germany, in general 5,531 turkeys [3,131 male and 2,400 female] of the British United Turkeys 6 strain from 46 flocks, were examined to that effect. Prevalence and severity increased within the duration of stay in the stable, and the prevalence was higher (P < 0.001) during the second visit between d 22 to 35 (factor: 0.94). Therefore, 27.3% (d 3 to 5; male/female: 39.1/25.0%) and 63.3% (d 22 to 35: 61.3/65.7%) of the examined poults had alterations of the foot pads, such as hyperkeratosis (d 3 to 5: 20.4/14.2%; d 22 to 35: 17.6/17.1%), high-grade hyperkeratosis with adhesive dirt (d 3 to 5: 8.7/10.7%; d 22 to 35: 29.2/39.3%), and epithelial necrosis (d 3 to 5: 0.1/0.1%; d 22 to 35: 14.6/9.3%). Female poults showed a higher risk (P < 0.001) of developing food pad alterations (factor: 0.76) than male poults. Male poults developed a higher percentage of epithelial necrosis than hens shortly before relocation. A higher stocking density during the very early rearing phase (d 3 to 5) led to a worse foot pad health status (P < 0.001). Because even mild alterations in the foot pad condition can be indicators for suboptimal design of the rearing environment and are to be seen as a pre-state for severe cases of foot pad dermatitis, it is important to set the main focus on the early rearing phase.
    Poultry Science 05/2013; 92(5):1171-6.
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    ABSTRACT: Traditionally, the bacteriological examination of mastitis milk samples is performed by culture followed by biochemical tests on the cultured bacteria to allow identification of the causative pathogen. Depending on the species involved, this classic identification is time-consuming compared to other techniques such as fluorescence in situ hybridization (FISH), a culture-independent method that utilizes oligonucleotides (labeled with a fluorophore) that are specific to a string of target DNA/RNA. In the current study, the applicability of FISH was evaluated for the detection of mastitis pathogens directly in milk samples. To remove interfering lipids and proteins from mastitis milk samples prior to FISH, a previously published enzymatic treatment with savinase was evaluated. FISH was performed using oligonucleotides specific for Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, and Trueperella (Arcanobacterium) pyogenes. The enzymatic pretreatment and the sensitivity of FISH were evaluated using spiked whole milk samples and mastitis milk samples with bacterial loads of less than 10(3) up to 10(8) colony-forming units (CFU)/ml. Bacteria were reliably detected in milk samples with bacterial numbers of 10(6) CFU/ml or higher. However, bacteria present in numbers below 10(6) CFU/ml were not detectable in all cases. The ability of FISH to identify mastitis-causing pathogens directly in milk samples, and therefore earlier than classical culture methods, can supplement the classic diagnostic procedures for mastitis milk samples.
    Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 04/2013;
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    ABSTRACT: Although many aspects of ovarian differentiation have been established, comparatively little is known about prenatal follicle formation and differentiation of bovine ovaries. The objective of this investigation was to study the role of the surface epithelium during the development of germ cell nests, germ cell cords and follicle formation in the fetal bovine ovary. Associated important proliferation and apoptotic features were further investigated. Additionally, the expression pattern of the S100 protein was detected. A strong increase of mitotic figures was detected in the surface epithelium, germ cell nests and germ cell cords of ovaries with a crown-rump length (CRL) of 13.0-58.0 cm. Oocytes were positively stained with S100 in bovine ovaries from fetuses with a CRL of 21.0 cm. The staining intensity enhanced parallel to increasing oocyte and follicle sizes during the ovary development. In later stages, a strong staining for S100 was observed in healthy oocytes in contradistinction to atretic oocytes where no expression of the S100 protein could be found. In conclusion, increasing mitosis index of surface epithelium cells, as well as oogonia directly beneath the surface epithelium, in combination with open surface connection during stages from a CRL of 11.0-94.0 cm of bovine fetal ovaries could play an important role in the period of time of ongoing folliculogenesis and derivation of granulosa cells. Additionally, S100-positive oocytes in primordial and later follicle stages joined by a high rate of Ki67-positive index in surrounding granulosa cells indicate that in the oocytes the S100 protein can perhaps be a useful marker for intact oocytes in bovine ovaries.
    Sexual Development 04/2013;
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    ABSTRACT: The nonhemolytic enterotoxin (Nhe) is known as a major pathogenicity factor for the diarrheal type of food poisoning caused by Bacillus cereus. The Nhe complex consists of NheA, NheB and NheC, all of them required to reach maximum cytotoxicity following a specific binding order on cell membranes. Here we show that complexes, formed between NheB and NheC under natural conditions before targeting the host cells, are essential for toxicity in Vero cells. To enable detection of NheC and its interaction with NheB, monoclonal antibodies against NheC were established and characterized. The antibodies allowed detection of recombinant NheC in a sandwich immunoassay at levels below 10 ng ml(-1), but no or only minor amounts of NheC were detectable in natural culture supernatants of B. cereus strains. When NheB- and NheC-specific monoclonal antibodies were combined in a sandwich immunoassay, complexes between NheB and NheC could be demonstrated. The level of these complexes was directly correlated with the relative concentrations of NheB and NheC. Toxicity, however, showed a bell-shaped dose-response curve with a plateau at ratios of NheB and NheC between 50:1 and 5:1. Both lower and higher ratios between NheB and NheC strongly reduced cytotoxicity. When the ratio approached an equimolar ratio, complex formation reached its maximum resulting in decreased binding of NheB to Vero cells. These data indicate that a defined level of NheB-NheC complexes as well as a sufficient amount of free NheB is necessary for efficient cell binding and toxicity. Altogether, the results of this study provide evidence that the interaction of NheB and NheC is a balanced process, necessary to induce, but also able to limit the toxic action of Nhe.
    PLoS ONE 01/2013; 8(4):e63104.
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    ABSTRACT: A high proportion of bovine oocytes fertilized in vitro fail to develop beyond the first 4 cleavage cycles. The first mitotic division of the zygote and proper segregation of chromosomes and cytoplasmic components seems to be a particularly delicate task. Notably, zygotes cleaving with a delay of only a few hours seem to have a very low chance of developing to the blastocyst stage. But what exactly goes wrong, how often, and why? To answer such questions we have to visualize in greater detail basic structures and processes such as the sperm aster, DNA replication, migration and apposition of the 2 pronuclei, synchronous chromosome condensation and breakdown of the nuclear envelopes, assembly of the first mitotic spindle and chromosome congression, anaphase, and cytokinesis. Oocytes fertilized in vitro were fixed at different time points around the first cleavage and stained for DNA, Ser10-phosphorylated histone H3, microtubules, and microfilaments. Zygotes were imaged in toto by recording confocal serial sections at 1-µm intervals using a 40× objective (NA=1.3). Details were recorded with high spatial sampling densities (pixel size 50×50nm, z-step size of 200nm) close to the Nyquist criterion and restored by maximum likelihood estimation deconvolution using the real point spread function. We present a series of 3-D confocal images captured at different stages of the first cleavage. The images reveal new insights into the formation, structure, and function of the first mitotic spindle and the occurrence of spindle aberrations, irregular chromosome segregation, and abnormal cytokinesis. The microscopic findings guide us to candidate proteins for localization analyses and functional studies based on 3-D fluorescence live-cell imaging of zygotes and early embryos.
    Reproduction Fertility and Development 12/2012; 25(1):199-200.
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    ABSTRACT: Hepatitis E virus (HEV) and Salmonella bacteria are zoonotic pathogens that can be acquired by foodborne transmission because food animals, for example pigs, are recognized as a reservoir. The objectives of this study were to determine the seroprevalence of anti-HEV immunoglobulin G (IgG) and anti-Salmonella antibodies from healthy pigs at slaughter in Switzerland, a country with a good health status of pig herds (e.g., eradication of enzootic pneumonia) compared with those of many countries in the European Union, and a rate of importation of live pigs that is very low (<1%). Based on pooled (diaphragm muscles from 3 to 5 animals per producer) meat juice samples, 120 (60%) of 200 and 8 (4%) of 200 samples were positive for anti-HEV IgG and anti-Salmonella antibodies, respectively. HEV seems to be highly prevalent among fattening pigs in Switzerland, whereas the low seroprevalence of anti-Salmonella IgG has not changed in recent years.
    Journal of food protection 08/2012; 75(8):1483-5.
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