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.

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.

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.

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.

Heartwater is a tick borne disease that affects ruminants and wild animals in Africa south of the Sahara. It is caused by Ehrlichia ruminantium and transmitted by the tick Amblyomma hebraeum. The protocols currently used to detect heartwater take several days to complete. Here, we describe the development of a pCS20 quantitative real-time PCRTaqMan probe assay to detect E. ruminantium in livestock blood and ticks from the field. The assay is based on the conserved pCS20 gene region of E. ruminantium that contains two overlapping genes, rnc and ctaG [Collins, N.E., Liebenberg, J., De Villiers, E.P., Brayton, K.A., Louw, E., Pretorius, A., Faber, F.E., Van Heerden, H., Josemans, A., Van Kleef, M., Steyn, H.C., Van Strijp, M.F., Zweygarth, E., Jongejan, F., Maillard, J.C., Berthier, D., Botha, M., Joubert, F., Corton, C.H., Thomson, N.R., Allsopp, M.T., Allsopp, B.A., 2005. The genome of the heartwater agent Ehrlichia ruminantium contains multiple tandem repeats of actively variable copy number. PNAS 102, 838–843]. The pCS20 quantitative real-time PCRTaqMan probe was compared to the currently used pCS20 PCR and PCR/32P-probe test with regards to sensitivity, specificity and the ability to detect DNA in field samples and in blood from experimentally infected sheep. This investigation showed that the pCS20 quantitative real-time PCRTaqMan probe was the most sensitive assay detecting seven copies of DNA/ml of cell culture. All three assays, however, cross react with Ehrlichia canis and Ehrlichia chaffeensis. The pCS20 real-time PCR detected significantly more positive field samples. Both the PCR and pCS20 real-time PCR could only detect E. ruminantium parasites in the blood of experimentally infected sheep during the febrile reaction. The PCR/32P-probe assay, however, detected the parasite DNA 1 day before and during the febrile reaction. Thus, because this new quantitative pCS20 real-time PCRTaqMan probe assay was the most sensitive and can be performed within 2 h it is an effective assay for epidemiological surveillance and monitoring of infected animals.

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.