Biomedical subjects

C M Davies

Publications and source records attributed to C M Davies.

At least 19 recordsLinked to original sources

PubMed2006

A comparison of non-radioactive methods for assessing viability in ex vivo cultured cancellous bone: technical note.

Biocompatibility studies are carried out either in two dimensional monolayer culture or in animal studies. Bone organ cultures are therefore required in order to reduce the number of animal studies performed, while at the same time ensuring a more natural environment than that provided by monolayer culture of isolated cells. Due to the three dimensional nature of bone explants, assays that determine the distribution of viable cells are required, however dense mineralised bone is not easily penetrated by soluble factors. We sought to compare a number of non-radioactive viability methods in order to assess their suitability for use with cancellous bone. Fluorescent live/dead staining, MTT activity and lactate dehydrogenase detection were all investigated on either whole bone explants (9.5 mm in diameter, 5 mm high) or on sections of explants. All these assays are routinely used in 2 dimensional cell culture systems, yet each required modifications to be suitable for use with cancellous bone. Factors such as penetration of reagent, incubation time, assay temperature and ease of determining viable cells were all compared. It was demonstrated that penetration of the reagents into whole cores was a major problem which easily led to artefacts that could be overcome by preparing 250 mum unfixed sections. Fluorescent live/dead staining had extra complications caused by the autofluorescence of the bone generating a high signal to noise ratio, making assessment of osteocyte viability impossible. MTT staining was difficult to interpret due to the punctate nature of the stain. We found that lactate dehydrogenase staining of 250 mum thick unfixed sections led to excellent viability determination of osteocytes within the mineralised matrix. It also maintained marrow structure and enabled marrow viability to be assessed as a factor of volume occupied by viable marrow. Decreasing the viscosity of the LDH assay solution used in published methods led to a greatly improved penetration into the calcified matrix. Quantification of thick sections is aided by using the autofluorescence of the bone to highlight the darkly stained osteocytes against the fluorescing bone.

Aged↗

PubMed2006

Mechanically loaded ex vivo bone culture system 'Zetos': systems and culture preparation.

This paper introduces the culture preparation of ovine, bovine and human cancellous bone cores to be used in an explants model Zetos. The three dimensional (3D) bone cores were prepared and evaluated for all three animals. Bone cells in vivo constantly interact with each other, migratory cells, surrounding extracellular matrix (ECM) and interstitial fluid in a microenvironment, which continuously responds to various endogenous and exogenous stimuli. The Zetos system was designed to culture and mechanically load viable cancellous bone explants in their near natural microenvironment. This 3D ex vivo system bridges the current gap between in vitro and in vivo methods. One aim of this work was to compare the macro and micro-architecture of ovine, bovine and human cancellous bone tissue in preparation for culture within the Zetos system in order to determine the optimal source of experimental material. A second aim was to optimise the preparations of the bone cores as well as develop techniques involved during tissue maintenance. Bone core response was visualised using histological and immunohistochemical methods. The results demonstrate that cancellous bone explants vary greatly in trabecular density and bone volume depending on species, age and location. Sheep and human samples displayed the greatest variation between bones cores when compared to bovine. Even cores taken from the same animal possessed very different characteristics. The histology demonstrated normal bone and cell structure after the core preparation. Immunohistochemistry results demonstrated antigen retention after preparation methods.

Aged↗

PubMed2006

Soil inactivation of DNA viruses in septic seepage.

AIMS: To generate field-relevant inactivation data for incorporation into models to predict the likelihood of viral contamination of surface waters by septic seepage. METHODS AND RESULTS: Inactivation rates were determined for PRD1 bacteriophage and Adenovirus 2 in two catchment soils under a range of temperature, moisture and biotic status regimes. Inactivation rates presented for both viruses were significantly different at different temperatures and in different soil types (alpha = 0.05). Soil moisture generally did not significantly affect virus inactivation rate. Biotic status significantly affected inactivation rates of PRD1 in the loam soil but not the clay-loam soil. Adenovirus 2 was inactivated more rapidly in the loam soil than PRD1 bacteriophage. CONCLUSIONS: Virus inactivation rates incorporated into models should be appropriate for the climate/catchment in question with particular regard to soil type and temperature. Given that PRD1 is similar in size to adenoviruses, yet more conservative with regard to inactivation in soil, it may be a useful surrogate in studies of Adenovirus fate and transport. SIGNIFICANCE AND IMPACT OF THE STUDY: A better understanding of the factors that govern virus fate and transport in catchments would facilitate the design of barrier measures to prevent viral contamination of surface waters by septic seepage.

Adenoviridae↗

PubMed2006

Microbial exposure assessment of an urban recreational lake: a case study of the application of new risk-based guidelines.

New WHO and Australian guidelines promote a risk-management approach for minimising exposure to pathogens in recreational waters. Between 2003 and 2005, they were applied to Lake Parramatta (10 ha, 450 ML), a potential recreation site in Sydney, Australia. A three stage approach was developed involving (1) initial suitability assessment using historic data, (2) revised suitability assessment based on new data and (3) characterisation of hazardous (especially wet weather) events. Contrary to the stage 1 suitability classification, stage 2 baseline data indicated that during dry weather the lake had water quality sufficient for primary contact recreation (95th percentiles for enterococci = 19 MPN/100, n = 50) and the major pathogen source was wildfowl. Guideline principles provided a rationale for collecting microbiological and geographic data needed to understand local cycles of lake contamination/recovery. The concept of hazardous events was particularly useful. Studies of stormwater events led us to identify a transition point (> 10 mm rainfall in 24 h) where human-faecal pathogen risks increased and access needed to be controlled. Together baseline and event data yielded operational tools (i.e. event detection methods, action triggers, auditing criteria, remediation priorities) for minimising bather exposure.

Clostridium perfringens↗

PubMed2005

A rapid radiochemical bacterial bioassay to evaluate copper toxicity in freshwaters.

A rapid, highly sensitive bacterial bioassay to determine copper toxicity in freshwaters was developed based on the inhibition of cellular assimilation of radiolabeled glucose. The test used a copper-sensitive bacterium isolated from a freshwater stream. Employing sensitive radiochemical techniques enabled environmentally relevant concentrations of the test bacterium (10(5) cells mL(-1)) and a short incubation period (4 hours) to be used, which minimized the potential for changes in copper speciation during the test. The 4-hour median effective concentration (EC(50)) for inorganic copper at pH 7.5 in synthetic freshwater was 0.6 microg L(-1) (95% confidence limits 0.4 to 1.0 microg L(-1)). This compared well with chronic growth inhibition of this bacterium in minimal medium (48-hour EC(50) of 0.9 microg L(-1) [95% confidence limits 0.7 to 1.0 microg L(-1)]). MINEQL + software (Environmental Research Software) was used to calculate copper (II) ion concentrations in synthetic freshwater at pH 7.5, giving an EC(50) value of pCu(2+) 8.8. However, using nitrilotriacetic acid metal-ion buffers (Cu-NTA), 50% inhibition occurred at a pCu(2+) of 9.7, suggesting this bacterium was markedly more inhibited by copper in these Cu(2+)-buffered solutions. This may indicate that the Cu-NTA species was contributing to toxicity. The radiochemical bioassay was evaluated further using freshwater samples from both copper-impacted and pristine environments. Measured EC(50) values ranged from 3.4 to 34.0 microg L(-1)inorganic copper and were strongly correlated with dissolved organic carbon (DOC) concentrations (r = 0.88, p < 0.05).

Australia↗

PubMed2005

Environmental inactivation of Cryptosporidium oocysts in catchment soils.

AIMS: To generate field-relevant inactivation rates for Cryptosporidium oocysts in soil that may serve as parameter values in models to predict the terrestrial fate and transport of oocysts in catchments. METHODS AND RESULTS: The inactivation of Cryptosporidium oocysts in closed soil microcosms over time was monitored using fluorescence in situ hybridization (FISH) as an estimate of oocyst 'viability'. Inactivation rates for Cryptosporidium in two soils were determined under a range of temperature, moisture and biotic status regimes. Temperature and soil type emerged as significantly influential factors (P < 0.05) for Cryptosporidium inactivation. In particular, temperatures as high as 35 degrees C may result in enhanced inactivation. CONCLUSIONS: When modelling the fate of Cryptosporidium oocysts in catchment soils, the use of inactivation rates that are appropriate for the specific catchment climate and soil types is essential. FISH was considered cost-effective and appropriate for determining oocyst inactivation rates in soil. SIGNIFICANCE AND IMPACT OF THE STUDY: Previous models for predicting the fate of pathogens in catchments have either made nonvalidated assumptions regarding inactivation of Cryptosporidium in the terrestrial environment or have not considered it at all. Field-relevant inactivation data are presented, with significant implications for the management of catchments in warm temperate and tropical environments.

Animals↗

PubMed2005

Evidence for the existence of Cryptosporidium oocysts as single entities in surface runoff.

There is uncertainty whether Cryptosporidium oocysts attach to particles or to each other under ambient water conditions. Particle size distributions of Cryptosporidium oocyst suspensions were determined over a range of ionic strengths and pHs to determine under those environmental conditions that may promote oocyst aggregation. Cryptosporidium oocysts were shown to only aggregate in high ionic strength solutions (>0.45 M) and remain largely as single entities at ionic strengths and pHs that were likely to be encountered in surface runoff. Similarly, in loam soil suspensions, rather than attaching to the soil particles the majority of oocysts also remained as single entities. Overall, oocysts are expected to remain largely unattached to either themselves or soil particles in overland runoff. This has implications for pathogen transport and modelling since oocysts that are freely suspended are more likely to be transported in runoff to surface waters than if attached to more dense soil/faecal particles.

Animals↗

PubMed2003

Immunohistochemistry of matrix markers in Technovit 9100 New-embedded undecalcified bone sections.

Trabecular bone is routinely analysed by histomorphological-histometrical and immunohistochemical techniques as means of assessing the differentiation status of bone deposition and growth. Currently few embedding resins exist for which both morphological and immunohistochemical analyses can be performed on mineralised tissue. Paraffin, the standard embedding medium for bone enzyme and immunohistochemistry, can only be used on demineralised tissue, but then trabecular structure may be badly preserved. Methyl methacrylate (MMA), the resin of choice for undecalcified bone histology can only be used for bone immunohistochemistry if the usual, highly exothermic polymerisation procedure is avoided which destroys tissue antigenicity. Consequently, most current practices involve cutting samples in half to be processed in separate resins when more than one type of analysis is required. Technovit 9100 New is a low temperature MMA embedding system that is purported to significantly improve tissue antigenicity preservation allowing polymerisation at -20 degrees C. In this study, Technovit 9100 New-embedded undecalcified trabecular bone samples (adult human, young bovine and ovine) yielded immunolabelling with several bone matrix markers and preserved morphological features in 7 microm sections when stained with Masson-Goldner, von Kossa, or toluidine blue. Bone samples from all resins used were immunolabelled with antibodies against osteocalcin, alkaline phosphatase, osteopontin, osteonectin, bone sialoprotein and procollagen type I amino-terminal propeptide. Technovit-embedded bone yielded more reliable immunolabelling of the matrix proteins when compared with heat or cold-cured LR White or standard embedded MMA samples. Technovit 9100 New provided better routine histology than LR White, and was comparable to MMA. Results demonstrated that Technovit 9100 New can be used as a low-temperature acrylic resin embedding method for routine undecalcified bone histology, as well as for immunohistochemistry.

Acrylic Resins↗

PubMed2003

Occurrence of coliphages in urban stormwater and their fate in stormwater management systems.

AIMS: To investigate the occurrence of coliphages in, and their removal from, urban stormwater. METHODS AND RESULTS: Inflow and outflow concentrations of somatic and f-specific RNA coliphages to two stormwater treatment systems were determined on 21 occasions over a period of 5 months. Somatic coliphages were detected in 19 (90%) of the constructed wetland inlet samples, 13 (62%) of the pond inlet samples, and less frequently at the outlets of the two systems. F-specific RNA coliphages were detected at the inlets but only occasionally at the pond outlet. Somatic coliphages were found to attach preferentially to particles <5 microm in size and persisted in the sediments of the two systems. CONCLUSIONS: Treatment systems providing conditions that are conducive to the settlement of fine particles may effectively remove sediment-bound coliphages and, therefore, possibly enteric viruses from stormwater. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will aid the design of systems for effective removal of viral contaminants from urban stormwater.

Coliphages↗

PubMed2003

Bacterial source tracking and shellfish contamination in a coastal catchment.

Introduced pathogens from faecal material can make their way into the aquatic environment from a number of catchment sources. These sources typically include sewage outfalls, seepage from septic tanks, and urban and agricultural runoff. Shellfish as filter feeders are particularly susceptible to contamination in faecally contaminated waters and a range of microbiological indicators have been developed to assess the levels of contamination and likely risks to public health (Hackney and Pierson, 1994). This paper outlines the application of bacterial source tracking (BST) in a shellfish growing area in part of the Port Stephens estuary along the NSW north coast. The approach is based on the premise that bacterial isolates from different faecal sources will have significantly different resistance patterns to the battery of antibiotics and concentrations tested. Faecal streptococci (FS) were isolated from several possible faecal sources: beef and dairy cattle, chickens and humans. The resistance patterns of these isolates to four different concentrations of four antibiotics were compared to those of FS isolates obtained from samples collected upstream and in the vicinity of the oyster leases. Discriminant analysis was performed using the patterns from the known source isolates and the rate of correct classification was determined for each source. The predictive function of discriminant analysis was then used to determine the most probable source of each of the unknown isolates from Tilligerry Creek, the drainage channels to the estuary, and the shellfish leases. Preliminary results are presented here and suggest that there is no single significant source of faecal contamination, rather there are contributions from a range of sources. The findings may have implications for the ways in which land use activities and catchments are managed in similar estuarine locations with a shellfish industry.

Agriculture↗

PubMed2002

Mixed strain schistosome infections of snails and the evolution of parasite virulence.

Mathematical models often propose that within-host competition between parasites can be a major factor in the evolution of increased parasite virulence. Kin selection predicts that as the coefficient of relatedness between infecting parasites decreases, the benefits of competition to individual genotypes increases. Thus where parasites can adjust their behaviour in response to current conditions, higher virulence is predicted in multiple genotype infections. There is limited experimental data, however, regarding the effects of mixed strain infections on host and parasite fitness. We investigated, for a snail-schistosome system, whether a conditional increase in replication rates occurred in mixed genotype infections and resulted in increased virulence. Four groups of Biomphalaria glabrata snails were exposed to 1 or 2 laboratory strains of Schistosoma mansoni. Mixed genotype infections were observed to be more virulent than single genotype infections, in terms of reductions in host reproductive success and survival. Parasite reproductive rate was also increased in mixed strain groups. Reduced host reproductive success was suggested to be directly due to the genetic heterogeneity of the parasitic infections resulting in increased host defence costs. Reduced host survival was consistent with an adaptive conditional parasite response.

Animals↗

PubMed2001

Trade-offs in the evolution of virulence in an indirectly transmitted macroparasite.

The adaptive trade-off theory for the evolution and maintenance of parasite virulence requires that virulence be genetically correlated with other fitness characteristics of the parasite. Many theoretical models rely on a positive correlation between virulence and transmissibility. They assume that high parasite replication rates are associated with a high probability of transmission (and, hence, increased parasite fitness), but also with high levels of damage to the host (high virulence). Schistosomes are macroparasites with an indirect life cycle involving a mammalian and a molluscan host. Here we demonstrate, through the development of five substrains, a genetic basis for schistosome virulence. We used these substrains further in order to investigate the presence of parasite fitness traits that were genetically correlated with virulence. High virulence in the (mouse) definitive host was, as predicted, positively correlated with parasite replication. In contrast, in the (snail) intermediate host high virulence was associated with low parasite replication rates. Variation in infectivity to and parasite replication in the definitive host was suggested as a compensating mechanism for the maintenance of virulence in the snail host. This is the first report of a trade-off in parasite reproductive success across hosts in an indirectly transmitted macroparasite.

Adaptation, Physiological↗

PubMed2001

Coevolution and compatibility in the snail-schistosome system.

In stark contrast to the huge body of theoretical work on the importance of hosts and parasites as selective agents acting on each other, until recently, little systematic empirical investigation of this issue has been attempted. Research on snail-schistosome interactions have, therefore, the potential for making an important contribution to the study of coevolution or reciprocal adaptation. This may be particularly pertinent since snail-schistosomes represent an indirectly transmitted macroparasite system, so often overlooked amongst both theoretical and empirical studies. Here we review ideas and experiments on snail-schistosome interactions, with particular emphasis on those that may have relevance to the potential coevolution between host resistance and parasite infectivity and virulence. We commence with an introduction and definition of the general concepts, before going into detail of some specific studies to illustrate these: evidence of snail-schistosome coevolutionary process in the field; evidence of coevolutionary processes in the laboratory; a general assessment of the applicability of coevolutionary models in snail-schistosome interactions; and finishing with a section on conclusions and areas for further study.

Animals↗

PubMed2001

Population genetics of the schistosome intermediate host Biomphalaria pfeifferi in the Zimbabwean highveld: implications for co-evolutionary theory.

Co-evolutionary theory proposes that polymorphisms in co-evolved traits may be maintained through differential selection in spatially heterogeneous environments. Spatial heterogeneity of the schistosome intermediate host, Biomphalaria pfeifferi, was investigated here, using RAPD markers. Overall, 256 individuals, collected at 32 sites on 13 rivers in the Chiweshe region of Zimbabwe, were analysed. Significant genetic differentiation was demonstrated, both between populations from the different rivers and between populations collected at different sites on each of several of the rivers investigated. However, the presence of spatial differentiation between populations from individual rivers varied with river type. It was not apparent in permanent, deep, fast-flowing rivers, where high levels of migration, through passive dispersal along the rivers, are likely. The snails collected from shallow, semi-permanent rivers not only showed relatively high levels of population subdivision but also high levels of within-site genetic diversity, consistent with the existence of 'co-evolutionary hot-spots' where schistosome infection may be prevalent. These results are discussed with reference to the population biology of B. pfeifferi and to host-parasite co-evolution.

Analysis of Variance↗

PubMed2001

Spatio-temporal genetic variability in the schistosome intermediate host Biomphalaria pfeifferi.

Genetic variation among temporally and spatially defined populations of the schistosome intermediate host, Biomphalaria pfeifferi, was analysed using RAPD markers. Snails were collected from each of eight sites that were 1-20 km apart, on four occasions, over a 2-year period. High levels of genetic variability were found within populations, between populations and between collections. Spatial differentiation and intrapopulation similarity were highest during periods of drought and were lowest following heavy rainfall. Temporal variability, consistent with founder effects, genetic drift and/or differential parasite pressure, was of similar or greater magnitude to that of the spatial variability. The results not only demonstrate that spatio-temporal sampling is necessary to understand the population genetics of short-lived species in variable habitats, but also provide empirical data to support current theoretical models of host-parasite co-evolution.

Analysis of Variance↗

PubMed2001

Stormwater treatment: do constructed wetlands yield improved pollutant management performance over a detention pond system?

Constructed wetland systems have been proposed as representing an improved ecotechnological option over detention basins, in terms of their abilities to reduce stormwater bacterial and nutrient loads to receiving waters. Concentrations of microbial and pollutants were determined in inflow and outflow samples collected from each type of system. Removal efficiencies for the wetland although higher than for the pond, were lower than some previously reported values for the treatment of municipal wastewater by constructed wetlands. Performance of a number of constructed wetland systems for stormwater treatment is evaluated considering the functional components of the systems.

Ecosystem↗

PubMed2000

The fate of stormwater-associated bacteria in constructed wetland and water pollution control pond systems.

The performances of a constructed wetland and a water pollution control pond were compared in terms of their abilities to reduce stormwater bacterial loads to recreational waters. Concentrations of thermotolerant coliforms, enterococci and heterotrophic bacteria were determined in inflow and outflow samples collected from each system over a 6-month period. Bacterial removal was significantly less effective in the water pollution control pond than in the constructed wetland. This was attributed to the inability of the pond system to retain the fine clay particles (< 2 microm) to which the bacteria were predominantly adsorbed. Sediment microcosm survival studies showed that the persistence of thermotolerant coliforms was greater in the pond sediments than in the wetland sediments, and that predation was a major factor influencing bacterial survival. The key to greater bacterial longevity in the pond sediments appeared to be the adsorption of bacteria to fine particles, which protected them from predators. These observations may significantly affect the choice of treatment system for effective stormwater management.

Bacteria↗

PubMed2000

An evaluation of potential interferences in a fluorimetric assay for the rapid detection of thermotolerant coliforms in sewage.

A 1-h fluorimetric assay of beta-D-galactosidase activity was evaluated for determining thermotolerant coliforms (TTC) in sewage samples. Above TTC concentrations of 2.3 x 103 colony-forming units (cfu) 100 ml-1, the assay response was related to TTC concentration. However, below this concentration, a large background signal was observed which was independent of TTC concentration. A separation scheme involving various filtration treatments and additions of a beta-D-galactosidase inhibitor was devised and used to quantify the sources of this anomalous assay response. The interferences encountered were largely due to the presence in sewage of non-specific cell-free enzymes or other cell-free substances that were capable of hydrolysing the fluorogenic substrate. Despite this apparent limitation, the fluorimetric enzyme assay has potential as an 'early warning' indicator of treatment process failure and gross sewage contamination and leakage in situations where TTC concentrations exceed 2.3 x 103 cfu 100 ml-1

Artifacts↗