In these studies we evaluated specific environmental stimuli relevant to Salmonella virulence and physiology in the gastrointestinal tract of chickens. Results from Salmonella growth in steady state, glucose-limiting continuous culture (CC) indicated that the optimal growth condition was observed between 0.05 h-1 and 0.27 h-1 dilution rates (D). Cell protein concentrations increased proportionally with an increase in D at each steady state, but after D 0.27 h-1 there was a reduction in the cell protein concentrations as the D increased. Genetic responses generally indicated that the lowest D exhibited highest hilA relative expression. Relatively higher expression of hilA was largely observed at low D (low glucose) (0.0125 h-1, 0.025 h-1, 0.05 h-1). Salmonella incubated in CC at different pH shifts demonstrated that cell protein concentration, glucose utilization, Yield ATP and Acetate:Propionate ratios were influenced by an increase in pH (6.14 to 7.41). These parameters increased and decreased consistently with a corresponding increase and decrease in pH. Polymerase chain reaction-based denaturant gradient gel electrophoresis showed that the overall amplicon band patterns of microbial similarity have demonstrated that hens molted with Alfalfa (ALC+) diet were similar to the Full-Fed (FF+) treatment group. Additional, FF+ and ALC+ treatment groups exhibited a higher percentage similarity coefficient (>90%) than the feed deprived treatment group. Fermentation response from cecal inocula on feed substrates revealed that alfalfa based samples yielded consistently higher short chain fatty acid levels when compared to other feed substrates. Salmonella Enteritidis (SE) colonization in liver, spleen and ovaries was significantly (P < 0.05) higher in FW+ hens compared to ALC+ and FF+ treatments groups. A 4-fold (log10 1.29) reduction in SE colonization for ALC+ hens compared to feed withdrawal hens (FW+) (log10 5.12) SE colonization was observed. Relative expression of hilA in all treatment groups was significantly (P < 0.05) higher in FW+ compared to FF+ and ALC+ groups. hilA expression in FW+ hens was 3.2-, 4.2-, and 1.9-fold higher for Days 6, 11 and 12 respectively, when compared with to ALC+ hens. These results suggest that Salmonella virulence in the gastrointestinal ecology of chickens could be impacted by a combination of low nutrients availability and pH shifts.

In these studies we evaluated specific environmental stimuli relevant to Salmonella virulence and physiology in the gastrointestinal tract of chickens. Results from Salmonella growth in steady state, glucose-limiting continuous culture (CC) indicated that the optimal growth condition was observed between 0.05 h-1 and 0.27 h-1 dilution rates (D). Cell protein concentrations increased proportionally with an increase in D at each steady state, but after D 0.27 h-1 there was a reduction in the cell protein concentrations as the D increased. Genetic responses generally indicated that the lowest D exhibited highest hilA relative expression. Relatively higher expression of hilA was largely observed at low D (low glucose) (0.0125 h-1, 0.025 h-1, 0.05 h-1). Salmonella incubated in CC at different pH shifts demonstrated that cell protein concentration, glucose utilization, Yield ATP and Acetate:Propionate ratios were influenced by an increase in pH (6.14 to 7.41). These parameters increased and decreased consistently with a corresponding increase and decrease in pH. Polymerase chain reaction-based denaturant gradient gel electrophoresis showed that the overall amplicon band patterns of microbial similarity have demonstrated that hens molted with Alfalfa (ALC+) diet were similar to the Full-Fed (FF+) treatment group. Additional, FF+ and ALC+ treatment groups exhibited a higher percentage similarity coefficient (>90%) than the feed deprived treatment group. Fermentation response from cecal inocula on feed substrates revealed that alfalfa based samples yielded consistently higher short chain fatty acid levels when compared to other feed substrates. Salmonella Enteritidis (SE) colonization in liver, spleen and ovaries was significantly (P < 0.05) higher in FW+ hens compared to ALC+ and FF+ treatments groups. A 4-fold (log10 1.29) reduction in SE colonization for ALC+ hens compared to feed withdrawal hens (FW+) (log10 5.12) SE colonization was observed. Relative expression of hilA in all treatment groups was significantly (P < 0.05) higher in FW+ compared to FF+ and ALC+ groups. hilA expression in FW+ hens was 3.2-, 4.2-, and 1.9-fold higher for Days 6, 11 and 12 respectively, when compared with to ALC+ hens. These results suggest that Salmonella virulence in the gastrointestinal ecology of chickens could be impacted by a combination of low nutrients availability and pH shifts.

Invasive Zygomykosen verzeichnen in den letzten Jahren eine steigende Inzidenz, insbesondere im Risikokollektiv immunsupprimierter Patienten. Aufgrund des häufig letalen Verlaufs dieser Infektionen ist eine rasche, korrekte Diagnosestellung essentiell, um rechtzeitig eine adäquate Therapie einzuleiten. Jedoch sieht sich die konventionelle, mikrobiologische Diagnostik mit vielen Problemen konfrontiert, so dass molekularbiologische Nachweisverfahren zunehmend in den Fokus der Aufmerksamkeit rücken. Eine zuverlässige, mit relativ geringem Zeit- und Kostenaufwand praktizierbare Methode stellt in diesem Zusammenhang die Real-time-PCR dar, deren Aussagekraft durch anschließende Speziesidentifizierung mittels Sequenzierung noch verstärkt werden kann. Aus diesem Grund wurden im Rahmen dieser Arbeit 3 PCR-Assays entwickelt und deren Sensitivität, Spezifität und klinische Anwendbarkeit evaluiert. Alle 3 Systeme nutzten Multi-copy-Gene des ribosomalen Operons der Zygomyzeten als Target und erwiesen sich als zuverlässige Werkzeuge zur Amplifikation fungaler DNA. Sie wurde sowohl an Pilzkulturen, als auch an klinischen Proben und einem Quasi-Tiermodell mit Erfolg ausgetestet und werden möglicherweise in Zukunft der klinischen Routinediagnostik zur Verfügung stehen. Bedingt durch die Seltenheit invasiver Zygomykosen besteht in diesem Bereich noch ein großer Forschungsbedarf, auch, um die noch nicht optimale Therapie dieser Erkrankungen zu verbessern. Es bleibt daher zu hoffen, dass sich in absehbarer Zeit mehr Forschungsgruppen mit diesen Erregern beschäftigen, damit den schwer kranken Patienten eine echte Heilungschance geboten werden kann. / During the last years invasive zygomycosis register a rising incidence, in particular in the risk collective of immunosuppressed patients. On account of the often lethal course of these infections a fast, correct diagnosis is essential to initiate an adequate therapy on time. However, the conventional, microbiological diagnostics is confronted with many problems, so that molecular-biological methods to detect invasive mucormycosis get moor important. A reliable method requiring relatively low time is the real-time-PCR. The following sequencing of the amplicon allows the identification to the genus level. That's why 3 PCR-Assays were developed and their sensitivity, specificity and clinical applicability were evaluated. All 3 systems targeted multi copy genes of the ribosomal operon of the zygomycetes and turned out to be reliable tools for the amplification of fungal DNA. They were successfully tested in fungal cultures, as well as in clinical tests and a quasi animal model. In future they will possibly be available to the clinical routine diagnostics. Because of the rarity of invasive zycomycosis a lot of research is needed in this area, also to improve the therapy, frequently being insufficient. However, there remains hope that in future more research groups deal with these causes, so that a real healing chance can be offered to the seriously ill patients.

Abstract Legionella species and Chlamydia pneumoniae are a common cause of community-acquired pneumonia and occasional cause of hospital-acquired pneumonia. Significant mortality rates among the elderly and patients with severe underlying disease may occur as a result of infection with those pathogen. Diagnostic delay may also result in increased mortality. Therefore, nucleic acid amplification assays have been shown to be useful for the detection of Legionella.spp and Chlamydia pneumoniae. The genes that encode 16S ribosomal subunits and the macrophage infectivity potentiator (MIP) gene have been shown to contain signature sequences that are useful for the identification of L. pneumophila and a variety of other Legionella species. The pst-1 fragment is useful for identification of Chlamydia pneumoniae. Here we try to test clinical specimens by Real-time PCR assays to detect L.pneumophila and other Legionella species in the same tube, and detect Chlamydia pneumoniae by SYBR Green 1 reagent. By this method, these amplicons of 16S ribosomal subunits gene and MIP gene can be discriminated by different melting curve dependent on different Tm values. In this study, we detected more 5 and 6 patients in Legionella species and Chlamydia pneumoniae than conventional diagnostic tools. Hence, the Real-time PCR also demonstrated that it¡¦s a rapid and high sensitivity method in diagnosis of legionnaires¡¦ disease. In this study, it also demonstrated that Real-time PCR is effective in prediction of atypical pathogen infection.

No description available.

BACKGROUND:Cryptococcus gattii has been the cause of an ongoing outbreak starting in 1999 on Vancouver Island, British Columbia and spreading to mainland Canada and the US Pacific Northwest. In the course of the outbreak, C. gattii has been identified outside of its previously documented climate, habitat, and host disease. Genotyping of C. gattii is essential to understand the ecological and geographical expansion of this emerging pathogen.METHODS:We developed and validated a mismatch amplification mutation assay (MAMA) real-time PCR panel for genotyping C. gattii molecular types VGI-VGIV and VGII subtypes a,b,c. Subtype assays were designed based on whole-genome sequence of 20 C. gattii strains. Publically available multilocus sequence typing (MLST) data from a study of 202 strains was used for the molecular type (VGI-VGIV) assay design. All assays were validated across DNA from 112 strains of diverse international origin and sample types, including animal, environmental and human.RESULTS:Validation revealed each assay on the panel is 100% sensitive, specific and concordant with MLST. The assay panel can detect down to 0.5 picograms of template DNA.CONCLUSIONS:The (MAMA) real-time PCR panel for C. gattii accurately typed a collection of 112 diverse strains and demonstrated high sensitivity. This is a time and cost efficient method of genotyping C. gattii best suited for application in large-scale epidemiological studies.

The age structure of populations is an important and often unresolved factor in ecology and wildlife management. Parameters like onset of reproduction and senescence, reproductive success and survival rate are tightly correlated with age. Unfortunately, age information of wild animals is not easy to obtain, especially for birds, where few anatomical markers of age exist. Longitudinal age data from birds banded as chicks are rare, particularly in long lived species. Age estimation in such species would be extremely useful as their long life span typically indicates slow population growth and potentially the need for protection and conservation. Telomere length change has been suggested as a universal marker for ageing vertebrates and potentially other animals. This method, termed molecular ageing, is based on a shortening of telomeres with each cell division. In birds, the telomere length of erythrocytes has been reported to decline with age, as the founder cells (haematopoietic stem cells) divide to renew circulating red blood cells. I measured telomere length in kakapo, the world largest parrot and four other bird species (Buller’s albatross, kea, New Zealand robin and saddleback) using telomere restriction fragment analysis (TRF) to assess the potential for molecular ageing in these species. After providing an overview of methods to measure telomere length, I describe how one of them (TRF) measures telomere length by quantifying the size distribution of terminal restriction fragments using southern blot of in-gel hybridization (Chapter 2). Although TRF is currently the ‘gold standard’ to measure telomere length, it suffers from various technical problems that can compromise precision and accuracy of telomere length estimation. In addition, there are many variations of the protocol, complicating comparisons between publications. I focused on TRF analysis using a non-radioactive probe, because it does not require special precautions associated with handling and disposing of radioactive material and therefore is more suitable for ecology laboratories that typically do not have a strong molecular biology infrastructure. However, most of my findings can be applied to both, radioactive and nonradioactive TRF variants. I tested how sample storage, choice of restriction enzyme, gel Abstract II electrophoresis and choice of hybridization buffer can influence the results. Finally, I show how image analysis (e.g. background correction, gel calibration, formula to calculate telomere length and the analysis window) can not only change the magnitude of estimated telomere length, but also their correlation to each other. Based on these findings, I present and discuss an extensive list of methodological difficulties associated with TRF and present a protocol to obtain reliable and reproducible results. Using this optimized protocol, I then measured telomere length of 68 kakapo (Chapter 3). Almost half of the current kakapo population consists of birds that were captured as adults, hence only their minimum age is known (i.e. time from when they were found +5 years to reach adulthood). Although molecular ageing might not be able to predict chronological age accurately, as calibrated with minimum age of some birds, it should be able to compare relative age between birds. Recently, the oldest kakapo (Richard Henry) was found to show signs of reproductive senescence. The age (or telomere length) difference to Richard Henry could have been used to approximate the remaining reproductive time span for other birds. Unfortunately, there was no change of telomere length with age in cross sectional and longitudinal samples. Analysis of fitness data available for kakapo yielded correlations between telomere length and fledging success, but they were weak and disappeared when the most influential bird was excluded from analysis. The heavy management and small numbers of kakapo make conclusions about fitness and telomere length difficult and highly speculative. However, telomere length of mothers and their chicks were significantly correlated, a phenomena not previously observed in any bird. To test if the lack of telomere loss with age is specific to kakapo, I measured telomere length of one of its closest relatives: the kea (Chapter 4). Like kakapo, telomere length did not show any correlation with age. I then further assessed the usefulness of molecular ageing in birds using only chicks and very old birds to estimate the maximum TL range in an additional long lived (Buller’s albatross) and two shorter lived species (NZ robin and saddleback). In these Abstract III species, telomere length was on average higher in chicks than in adults. However, age matched individuals showed high variations in telomere length, such that age dependent and independent telomere length could not be distinguished. These data and published results from other bird species, coupled with the limitations of methodology I have identified (Chapter 2), indicate that molecular ageing does not work in most (if not all) birds in its current suggested form. Another way to measure telomere length is telomere Q-PCR, a real-time PCR based method. Measurement of the same kakapo samples with TRF and Q-PCR did not result in comparable results (Chapter 4). Through experimentation I found that differences in amplification efficiency between samples lead to unreliable estimation of telomere length using telomere Q-PCR. These differences were caused by inhibitors present in the samples. The problem of differential amplification efficiency in Q-PCR, while known, is largely ignored by the scientific community. Although some methods have been suggested to correct for differing efficiency, most of these introduce more error than they eliminate. I developed and applied an assay based on internal standard oligonucleotides that was able to corrected EDTA induced quantification errors of up to 70% with high precision and accuracy (Chapter 5). The method, however, failed when tested with other inhibitors commonly found in DNA samples extracted from blood (i.e. SDS, heparin, urea and FeCl3). PCR inhibition was highly selective in the probe-polymerase system I used, inhibiting amplification of genomic DNA, but not amplification of internal oligonucleotide or plasmid standards in the same reaction. Internal standards are a key feature of most diagnostic PCR assays to identify false negatives arising from amplification inhibition. The differential response to inhibition I identified greatly compromises the accuracy of these assays. Consequently, I strongly recommend that researchers using PCR assays with internal standards should verify that the target DNA and internal standard actually respond similarly to common inhibitors.

Neurturin (NTN) belongs to the glial cell-line derived neurotrophic factor (GDNF) family of growth factors. Both NTN and GDNF have been shown to potently prevent the degeneration of dopaminergic neuron in vitro and in vivo. The GDNF family receptor alpha 2 (GFRα-2) is the preferred receptor for NTN. In addition to the known full-length isoform (GFRα-2a), we have previously reported the isolation of two novel alternatively spliced isoforms (GFRα-2b and GFRα-2c). The expression levels of these isoforms have yet to be quantified and the functional properties determined. In this report, we have developed a real time polymerase chain reaction (PCR) using SYBR Green I to detect the expression levels of the three splice variants (GFRα-2a, GFRα-2b and GFRα-2c) in murine tissues. Both GFRα-2a and GFRα-2c were expressed at similar levels in all tissues examined. GFRα-2b was found to be 10 fold lower in expression. All three isoforms activated MAPK (ERK1/2) and Akt. Transcriptional profiling with DNA microarrays demonstrated that the spliced isoforms do not share similar profiles. In conclusion, we have now shown the expression levels of the spliced variants. All three isoforms are functional. However, each isoform appeared to have unique transcriptional profiles when activated. / Singapore-MIT Alliance (SMA)

Steroid hormones, such as estrogen, are produced in one tissue and carried through the blood stream to target tissues in which they bind to highly specific nuclear receptors and trigger changes in gene expression and metabolism. Industrial chemicals, such as bisphenol A and many agricultural chemicals, including permethrin and fervalerate, are known to have estrogenic potential and therefore are estrogen mimics. Widely used agricultural chemicals, Enable (fungicide) and Diazinon (insecticide), were evaluated to examine their toxicity and estrogenicity. MCF-7 cells, an estrogen-dependent human breast cancer line, were utilized for this purpose. MCF-7 cells were treated with 0.033-3.3 ppb (ng/ml) of Enable and 0.3-67 ppm of Diazinon and gene expression was compared to that in untreated cells. Microarray analysis showed down-regulation of eight genes and up-regulation of thirty four genes in cells treated with 3.3 ppb of Enable, compared to untreated cells. Similarly, in cells treated with 67 ppm of Diazinon, there were three genes down-regulated and twenty seven genes up-regulated. For both chemicals, specific genes were selected for special consideration. RT-PCR confirmed results obtained from analysis of the microarray. These studies were designed to provide base-line data on gene expression-altering capacity of specific chemicals and will allow assessment of the deleterious effects caused by exposure to the aforementioned chemicals.

ABSTRACT Two experiments were conducted to determine the effects of various forage to concentrate ratios on the rumen microbial ecosystem and rumen fermentation parameters using culture-independent methods. In the first experiment, cattle were fed either a high concentrate (HC) or a high concentrate without forage (HCNF) diet. Comparison of rumen fermentation parameters between these two diets showed that duration of time spent below pH 5.2 and rumen osmolality were higher for HCNF. Calculations using Simpson’s index showed a greater diversity of dominant species for HCNF than in HC based on 16S rRNA PCR-DGGE. Real-time real-time PCR showed populations of Fibrobacter succinogenes (P=0.01) were lower in HCNF than HC diets. Ruminococcus spp., F. succinogenes and Selenomonas ruminantium were present at higher (P≤0.05) concentrations in solid than in liquid digesta in both diets. The second experiment compared cattle as they adapted from a strictly forage to a concentrate diet, after which they were subject to an acidotic challenge and a recovery period (Forage, Mixed Forage, High Grain, Acidosis and Recovery). A total of 153,621 high-quality bacterial sequences were obtained from biopsied rumen epithelium, and 407,373 sequences from the solid and liquid phases of rumen contents. Only 14 epithelial genera representing >1.0% of the epimural population differed (P ≤ 0.05) among dietary treatments. However, clustering showed a closer relation in bacterial profiles for the Forage and Mixed Forage diets as compared to the High Grain, Acidosis and Recovery diets. Several epithelial identified genera including Atopobium, Desulfocurvus, Fervidicola, Lactobacillus and Olsenella increased as a result of acidosis. However, any changes in bacterial populations during the acidosis challenge were not sustained during the recovery period. This indicates a high level of stability within the rumen epimural community. An epithelial core microbiome was determined which explained 21% of the enumerable rumen population across all treatment samples. Cluster analysis of the solid and liquid phase rumen bacterial showed that these populations differed (P ≤ 0.10) between forage and grain-based diets. Rumen core microbiome analysis found 32 OTU’s representing 10 distinct bacterial taxa in whole rumen contents for all dietary treatments. Heifers that developed clinical acidosis vs the subclinical acidosis showed increases in the genera Acetitomaculum, Lactobacillus, Prevotella, and Streptococcus. Variation in microbial taxa as an effect of both treatment and animal was evident in the solid and liquid fractions of the rumen digesta. However, impacts of a dietary treatment were transient and despite an acidotic challenge, rumen microbiota were able to recover within a week of perturbation. The bacterial populations in the rumen are highly diverse as indicated by DGGE analysis and showed clear distinction between not only dietary treatments, individual animals, but also between epithelial, liquid and solid associated populations on the same diet. Molecular techniques provide an increased understanding of the impact of dietary change on the nature of rumen bacterial populations and conclusions derived using these techniques may not match those previously derived using traditional laboratory culturing techniques.