Morphologic and genomic characterisation of the koala Chlamydia pneumoniae strain

Chlamydia pneumoniae is a common human and animal pathogen associated with a wide range of upper and lower respiratory tract infections. In more recent years there has been increasing evidence to suggest a link between C. pneumoniae and chronic diseases in humans, including atherosclerosis, stroke and Alzheimer’s disease. C. pneumoniae human strains show little genetic variation, indicating that the human-derived strain originated from a common ancestor in the recent past. Despite extensive information on the genetics and morphology processes of the human strain, knowledge concerning many other hosts (including marsupials, amphibians, reptiles and equines) remains virtually unexplored. The koala (Phascolarctos cinereus) is a native Australian marsupial under threat due to habitat loss, predation and disease. Koalas are very susceptible to chlamydial infections, most commonly affecting the conjunctiva, urogenital tract and/or respiratory tract. To address this gap in the literature, the present study (i) provides a detailed description of the morphologic and genomic architecture of the C. pneumoniae koala (and human) strain, and shows that the koala strain is microscopically, developmentally and genetically distinct from the C. pneumoniae human strain, and (ii) examines the genetic relationship of geographically diverse C. pneumoniae isolates from human, marsupial, amphibian, reptilian and equine hosts, and identifies two distinct lineages that have arisen from animal-to-human cross species transmissions. Chapter One of this thesis explores the scientific problem and aims of this study, while Chapter Two provides a detailed literature review of the background in this field of work. Chapter Three, the first results chapter, describes the morphology and developmental stages of C. pneumoniae koala isolate LPCoLN, as revealed by fluorescence and transmission electron microscopy. The profile of this isolate, when cultured in HEp-2 human epithelial cells, was quite different to the human AR39 isolate. Koala LPCoLN inclusions were larger; the elementary bodies did not have the characteristic pear-shaped appearance, and the developmental cycle was completed within a shorter period of time (as confirmed by quantitative real-time PCR). These in vitro findings might reflect biological differences between koala LPCoLN and human AR39 in vivo. Chapter Four describes the complete genome sequence of the koala respiratory pathogen, C. pneumoniae LPCoLN. This is the first animal isolate of C. pneumoniae to be fully-sequenced. The genome sequence provides new insights into genomic ‘plasticity’ (organisation), evolution and biology of koala LPCoLN, relative to four complete C. pneumoniae human genomes (AR39, CWL029, J138 and TW183). Koala LPCoLN contains a plasmid that is not shared with any of the human isolates, there is evidence of gene loss in nucleotide salvage pathways, and there are 10 hot spot genomic regions of variation that were previously not identified in the C. pneumoniae human genomes. Sequence (partial-length) from a second, independent, wild koala isolate (EBB) at several gene loci confirmed that the koala LPCoLN isolate was representative of a koala C. pneumoniae strain. The combined sequence data provides evidence that the C. pneumoniae animal (koala LPCoLN) genome is ancestral to the C. pneumoniae human genomes and that human infections may have originated from zoonotic infections. Chapter Five examines key genome components of the five C. pneumoniae genomes in more detail. This analysis reveals genomic features that are shared by and/or contribute to the broad ecological adaptability and evolution of C. pneumoniae. This analysis resulted in the identification of 65 gene sequences for further analysis of intraspecific variation, and revealed some interesting differences, including fragmentation, truncation and gene decay (loss of redundant ancestral traits). This study provides valuable insights into metabolic diversity, adaptation and evolution of C. pneumoniae. Chapter Six utilises a subset of 23 target genes identified from the previous genomic comparisons and makes a significant contribution to our understanding of genetic variability among C. pneumoniae human (11) and animal (6 amphibian, 5 reptilian, 1 equine and 7 marsupial hosts) isolates. It has been shown that the animal isolates are genetically diverse, unlike the human isolates that are virtually clonal. More convincing evidence that C. pneumoniae originated in animals and recently (in the last few hundred thousand years) crossed host species to infect humans is provided in this study. It is proposed that two animal-to-human cross species events have occurred in the context of the results, one evident by the nearly clonal human genotype circulating in the world today, and the other by a more animal-like genotype apparent in Indigenous Australians. Taken together, these data indicate that the C. pneumoniae koala LPCoLN isolate has morphologic and genomic characteristics that are distinct from the human isolates. These differences may affect the survival and activity of the C. pneumoniae koala pathogen in its natural host, in vivo. This study, by utilising the genetic diversity of C. pneumoniae, identified new genetic markers for distinguishing human and animal isolates. However, not all C. pneumoniae isolates were genetically diverse; in fact, several isolates were highly conserved, if not identical in sequence (i.e. Australian marsupials) emphasising that at some stage in the evolution of this pathogen, there has been an adaptation/s to a particular host, providing some stability in the genome. The outcomes of this study by experimental and bioinformatic approaches have significantly enhanced our knowledge of the biology of this pathogen and will advance opportunities for the investigation of novel vaccine targets, antimicrobial therapy, or blocking of pathogenic pathways.

The design and evaluation of a mobile sensor/actuator network for autonomous animal control

This paper investigates a mobile, wireless sensor/actuator network application for use in the cattle breeding industry. Our goal is to prevent fighting between bulls in on-farm breeding paddocks by autonomously applying appropriate stimuli when one bull approaches another bull. This is an important application because fighting between high-value animals such as bulls during breeding seasons causes significant financial loss to producers. Furthermore, there are significant challenges in this type of application because it requires dynamic animal state estimation, real-time actuation and efficient mobile wireless transmissions. We designed and implemented an animal state estimation algorithm based on a state-machine mechanism for each animal. Autonomous actuation is performed based on the estimated states of an animal relative to other animals. A simple, yet effective, wireless communication model has been proposed and implemented to achieve high delivery rates in mobile environments. We evaluated the performance of our design by both simulations and field experiments, which demonstrated the effectiveness of our autonomous animal control system.

Transforming agriculture through pervasive wireless sensor networks

A large-scale, outdoor, pervasive computing system based on the Fleck hardware platform applies sensor network technology to farming. Comprising static and animal-borne mobile nodes, the system measures the state of a complex, dynamic system comprising climate, soil, pasture, and animals. This data supports prediction of the land's future state and improved management outcomes through closed-loop control. This article is part of a special issue, Building a Sensor-Rich World.

Animal behaviour understanding using wireless sensor networks

This paper presents research that is being conducted by the Commonwealth Scientific and Industrial Research Organisation (CSIRO) with the aim of investigating the use of wireless sensor networks for automated livestock monitoring and control. It is difficult to achieve practical and reliable cattle monitoring with current conventional technologies due to challenges such as large grazing areas of cattle, long time periods of data sampling, and constantly varying physical environments. Wireless sensor networks bring a new level of possibilities into this area with the potential for greatly increased spatial and temporal resolution of measurement data. CSIRO has created a wireless sensor platform for animal behaviour monitoring where we are able to observe and collect information of animals without significantly interfering with them. Based on such monitoring information, we can identify each animal's behaviour and activities successfully

Results from the farm

This paper reports on a large, long-term mobile wireless sensor network deployment. The trial was part of an animal study involving 45 animals. During the trial, 15 animals were equipped with wireless sensor nodes for a week. The paper discusses various issues with such a deployment including electronic design, software design, animal ethics clearance, logistics, and wearable computing equipment for animals. The paper also presents some preliminary analysis of the data obtained from the deployment, both from the perspective of network parameters and animal movement behavior.

Wireless adhoc sensor and actuator networks on the farm

Agriculture accounts for a significant portion of the GDP in most developed countries. However, managing farms, particularly largescale extensive farming systems, is hindered by lack of data and increasing shortage of labour. We have deployed a large heterogeneous sensor network on a working farm to explore sensor network applications that can address some of the issues identified above. Our network is solar powered and has been running for over 6 months. The current deployment consists of over 40 moisture sensors that provide soil moisture profiles at varying depths, weight sensors to compute the amount of food and water consumed by animals, electronic tag readers, up to 40 sensors that can be used to track animal movement (consisting of GPS, compass and accelerometers), and 20 sensor/actuators that can be used to apply different stimuli (audio, vibration and mild electric shock) to the animal. The static part of the network is designed for 24/7 operation and is linked to the Internet via a dedicated high-gain radio link, also solar powered. The initial goals of the deployment are to provide a testbed for sensor network research in programmability and data handling while also being a vital tool for scientists to study animal behavior. Our longer term aim is to create a management system that completely transforms the way farms are managed.

Animal Control - What constitutes a reliable cue to stop animal movement ?

Controlling free-ranging livestock requires low-stress cues to alter animal behaviour. Recently modulated sound and electric shock were demonstrated to be effective in controlling free-ranging cattle. In this study the behaviour of 60, 300 kg Belmont Red heifers were observed for behavioural changes when presented cues designed to impede their movement through an alley. The heifers were given an overnight drylot shrink off feed but not drinking water prior to being tested. Individual cattle were allowed to move down a 6.5 m wide alley towards a pen of peers and feed located 71 m from their point of release. Each animal was allowed to move through the alley unimpeded five times to establish a basal behavioural pattern. Animals were then randomly assigned to treatments consisting of sound plus shock, vibration plus shock, a visual cue plus shock, shock by itself and a control. The time each animal required to reach the pen of peers and feed was recorded. If the animal was prevented from reaching the pen of peers and feed by not penetrating through the cue barrier at set points along the alley for at least 60 sec the test was stopped and the animal was returned to peers located behind the release pen. Cues and shock were manually applied from a laptop while animals were observed from a 3.5 m tower located outside the alley. Electric shock, sound, vibration and Global Position System (GPS) hardware were housed in a neck collar. Results and implications will be discussed.

Dynamic virtual fences for controlling cows

A virtual fence is created by applying an aversive stimulus to an animal when it approaches a predefined boundary. It is implemented by a small animal-borne computer system with a GPS receiver. This approach allows the implementation of virtual paddocks inside a normal physically-fenced paddock. Since the fence lines are virtual they can be moved by programming to meet the needs of animal or land management. This approach enables us to consider animals as agents with natural mobility that are controllable and to apply a vast body of theory in motion planning. In this paper we describe a herd-animal simulator and physical experiments conducted on a small herd of 10 animals using a Smart Collar. The Smart Collar consists of a GPS, PDA, wireless networking and a sound amplifier. We describe a motion planning algorithm that can move a virtual paddock subject to landscape constraints which is suitable for mustering cows. We present simulation results and data from experiments with 8 cows equipped with Smart Collars.

Poster : Understanding interactions between autonomous animal control and temperament when cattle are subjected to virtual fencing applications

Virtual fencing has the potential to control grazing livestock. Understanding and refi ning the cues that can alter behaviour is an integral part of autonomous animal control. A series of tests have been completed to explore the relationship between temperament and control. Prior to exposure to virtual fencing control the animals were scored for temperament using fl ight speed and a sociability index using contact logging devices. The behavioural response of 30, Belmont Red steers were observed for behavioural changes when presented with cues prior to receiving an electrical stimulation. A control and four treatments designed to interrupt the animal’s movement down an alley were tested. The treatments consisted of sound plus electrical stimulation, vibration plus electrical stimulation, a visual cue plus electrical stimulation and electrical stimulation by itself. The treatments were randomly applied to each animal over fi ve consecutive trials. A control treatment in which no cues were applied was used to establish a basal behavioural pattern. A trial was considered completed after each animal had been retained behind the cue barrier for at least 60 sec. All cues and electrical stimulation were manually applied from a laptop located on a portable 3.5 m tower located immediately outside the alley. The electric stimulation consisted of 1.0 Kv of electricity. Electric stimulation, sound and vibration along with the Global Position System (GPS) hardware to autonomously record the animal’s path within the alley were recorded every second.

Immunogenicity and protective efficacy of an anti-Streptococcus pyogenes vaccine candidate in multiple animal species

Streptococcus pyogenes, also known as Group A Streptococcus (GAS) has been associated with a range of diseases from the mild pharyngitis and pyoderma to more severe invasive infections such as streptococcal toxic shock. GAS also causes a number of non-suppurative post-infectious diseases such as rheumatic fever, rheumatic heart disease and glomerulonephritis. The large extent of GAS disease burden necessitates the need for a prophylactic vaccine that could target the diverse GAS emm types circulating globally. Anti-GAS vaccine strategies have focused primarily on the GAS M-protein, an extracellular virulence factor anchored to GAS cell wall. As opposed to the hypervariable N-terminal region, the C-terminal portion of the protein is highly conserved among different GAS emm types and is the focus of a leading GAS vaccine candidate, J8-DT/alum. The vaccine candidate J8-DT/alum was shown to be immunogenic in mice, rabbits and the non-human primates, hamadryas baboons. Similar responses to J8-DT/alum were observed after subcutaneous and intramuscular immunization with J8-DT/alum, in mice and in rabbits. Further assessment of parameters that may influence the immunogenicity of J8-DT demonstrated that the immune responses were identical in male and female mice and the use of alum as an adjuvant in the vaccine formulation significantly increased its immunogenicity, resulting in a long-lived serum IgG response. Contrary to the previous findings, the data in this thesis indicates that a primary immunization with J8-DT/alum (50ƒÊg) followed by a single boost is sufficient to generate a robust immune response in mice. As expected, the IgG response to J8- DT/alum was a Th2 type response consisting predominantly of the isotype IgG1 accompanied by lower levels of IgG2a. Intramuscular vaccination of rabbits with J8-DT/alum demonstrated that an increase in the dose of J8-DT/alum up to 500ƒÊg does not have an impact on the serum IgG titers achieved. Similar to the immune response in mice, immunization with J8-DT/alum in baboons also established that a 60ƒÊg dose compared to either 30ƒÊg or 120ƒÊg was sufficient to generate a robust immune response. Interestingly, mucosal infection of naive baboons with a M1 GAS strain did not induce a J8-specific serum IgG response. As J8-DT/alum mediated protection has been previously reported to be due to the J8- specific antibody formed, the efficacy of J8-DT antibodies was determined in vitro and in vivo. In vitro opsonization and in vivo passive transfer confirmed the protective potential of J8-DT antibodies. A reduction in the bacterial burden after challenge with a bioluminescent M49 GAS strain in mice that were passively administered J8-DT IgG established that protection due to J8-DT was mediated by antibodies. The GAS burden in infected mice was monitored using bioluminescent imaging in addition to traditional CFU assays. Bioluminescent GAS strains including the ‘rheumatogenic’ M1 GAS could not be generated due to limitations with transformation of GAS, however, a M49 GAS strain was utilized during BLI. The M49 serotype is traditionally a ‘nephritogenic’ serotype associated with post-streptococcal glomerulonephritis. Anti- J8-DT antibodies now have been shown to be protective against multiple GAS strains such as M49 and M1. This study evaluated the immunogenicity of J8-DT/alum in different species of experimental animals in preparation for phase I human clinical trials and provided the ground work for the development of a rapid non-invasive assay for evaluation of vaccine candidates.

The influence of habitat heterogeneity on patterns of connectivity among rabbit populations in southern Queensland

Patterns of connectivity among local populations influence the dynamics of regional systems, but most ecological models have concentrated on explaining the effect of connectivity on local population structure using dynamic processes covering short spatial and temporal scales. In this study, a model was developed in an extended spatial system to examine the hypothesis that long term connectivity levels among local populations are influenced by the spatial distribution of resources and other habitat factors. The habitat heterogeneity model was applied to local wild rabbit populations in the semi-arid Mitchell region of southern central Queensland (the Eastern system). Species' specific population parameters which were appropriate for the rabbit in this region were used. The model predicted a wide range of long term connectivity levels among sites, ranging from the extreme isolation of some sites to relatively high interaction probabilities for others. The validity of model assumptions was assessed by regressing model output against independent population genetic data, and explained over 80% of the variation in the highly structured genetic data set. Furthermore, the model was robust, explaining a significant proportion of the variation in the genetic data over a wide range of parameters. The performance of the habitat heterogeneity model was further assessed by simulating the widely reported recent range expansion of the wild rabbit into the Mitchell region from the adjacent, panmictic Western rabbit population system. The model explained well the independently determined genetic characteristics of the Eastern system at different hierarchic levels, from site specific differences (for example, fixation of a single allele in the population at one site), to differences between population systems (absence of an allele in the Eastern system which is present in all Western system sites). The model therefore explained the past and long term processes which have led to the formation and maintenance of the highly structured Eastern rabbit population system. Most animals exhibit sex biased dispersal which may influence long term connectivity levels among local populations, and thus the dynamics of regional systems. When appropriate sex specific dispersal characteristics were used, the habitat heterogeneity model predicted substantially different interaction patterns between female-only and combined male and female dispersal scenarios. In the latter case, model output was validated using data from a bi-parentally inherited genetic marker. Again, the model explained over 80% of the variation in the genetic data. The fact that such a large proportion of variability is explained in two genetic data sets provides very good evidence that habitat heterogeneity influences long term connectivity levels among local rabbit populations in the Mitchell region for both males and females. The habitat heterogeneity model thus provides a powerful approach for understanding the large scale processes that shape regional population systems in general. Therefore the model has the potential to be useful as a tool to aid in the management of those systems, whether it be for pest management or conservation purposes.

Swept frequency biompedance analysis for the determination of body water compartments

Bioelectrical impedance analysis, (BIA), is a method of body composition analysis first investigated in 1962 which has recently received much attention by a number of research groups. The reasons for this recent interest are its advantages, (viz: inexpensive, non-invasive and portable) and also the increasing interest in the diagnostic value of body composition analysis. The concept utilised by BIA to predict body water volumes is the proportional relationship for a simple cylindrical conductor, (volume oc length2/resistance), which allows the volume to be predicted from the measured resistance and length. Most of the research to date has measured the body's resistance to the passage of a 50· kHz AC current to predict total body water, (TBW). Several research groups have investigated the application of AC currents at lower frequencies, (eg 5 kHz), to predict extracellular water, (ECW). However all research to date using BIA to predict body water volumes has used the impedance measured at a discrete frequency or frequencies. This thesis investigates the variation of impedance and phase of biological systems over a range of frequencies and describes the development of a swept frequency bioimpedance meter which measures impedance and phase at 496 frequencies ranging from 4 kHz to 1 MHz. The impedance of any biological system varies with the frequency of the applied current. The graph of reactance vs resistance yields a circular arc with the resistance decreasing with increasing frequency and reactance increasing from zero to a maximum then decreasing to zero. Computer programs were written to analyse the measured impedance spectrum and determine the impedance, Zc, at the characteristic frequency, (the frequency at which the reactance is a maximum). The fitted locus of the measured data was extrapolated to determine the resistance, Ro, at zero frequency; a value that cannot be measured directly using surface electrodes. The explanation of the theoretical basis for selecting these impedance values (Zc and Ro), to predict TBW and ECW is presented. Studies were conducted on a group of normal healthy animals, (n=42), in which TBW and ECW were determined by the gold standard of isotope dilution. The prediction quotients L2/Zc and L2/Ro, (L=length), yielded standard errors of 4.2% and 3.2% respectively, and were found to be significantly better than previously reported, empirically determined prediction quotients derived from measurements at a single frequency. The prediction equations established in this group of normal healthy animals were applied to a group of animals with abnormally low fluid levels, (n=20), and also to a group with an abnormal balance of extra-cellular to intracellular fluids, (n=20). In both cases the equations using L2/Zc and L2/Ro accurately and precisely predicted TBW and ECW. This demonstrated that the technique developed using multiple frequency bioelectrical impedance analysis, (MFBIA), can accurately predict both TBW and ECW in both normal and abnormal animals, (with standard errors of the estimate of 6% and 3% for TBW and ECW respectively). Isotope dilution techniques were used to determine TBW and ECW in a group of 60 healthy human subjects, (male. and female, aged between 18 and 45). Whole body impedance measurements were recorded on each subject using the MFBIA technique and the correlations between body water volumes, (TBW and ECW), and heighe/impedance, (for all measured frequencies), were compared. The prediction quotients H2/Zc and H2/Ro, (H=height), again yielded the highest correlation with TBW and ECW respectively with corresponding standard errors of 5.2% and 10%. The values of the correlation coefficients obtained in this study were very similar to those recently reported by others. It was also observed that in healthy human subjects the impedance measured at virtually any frequency yielded correlations not significantly different from those obtained from the MFBIA quotients. This phenomenon has been reported by other research groups and emphasises the need to validate the technique by investigating its application in one or more groups with abnormalities in fluid levels. The clinical application of MFBIA was trialled and its capability of detecting lymphoedema, (an excess of extracellular fluid), was investigated. The MFBIA technique was demonstrated to be significantly more sensitive, (P<.05), in detecting lymphoedema than the current technique of circumferential measurements. MFBIA was also shown to provide valuable information describing the changes in the quantity of muscle mass of the patient during the course of the treatment. The determination of body composition, (viz TBW and ECW), by MFBIA has been shown to be a significant improvement on previous bioelectrical impedance techniques. The merit of the MFBIA technique is evidenced in its accurate, precise and valid application in animal groups with a wide variation in body fluid volumes and balances. The multiple frequency bioelectrical impedance analysis technique developed in this study provides accurate and precise estimates of body composition, (viz TBW and ECW), regardless of the individual's state of health.

In vivo knockdown of the androgen receptor results in growth inhibition and regression of well-established, castration-resistant prostate tumours

Purpose: Progression to the castration-resistant state is the incurable and lethal end stage of prostate cancer, and there is strong evidence that androgen receptor (AR) still plays a central role in this process. We hypothesize that knocking down AR will have a major effect on inhibiting growth of castration-resistant tumors. Experimental Design: Castration-resistant C4-2 human prostate cancer cells stably expressing a tetracycline-inducible AR-targeted short hairpin RNA (shRNA) were generated to directly test the effects of AR knockdown in C4-2 human prostate cancer cells and tumors. Results:In vitro expression of AR shRNA resulted in decreased levels of AR mRNA and protein, decreased expression of prostate-specific antigen (PSA), reduced activation of the PSA-luciferase reporter, and growth inhibition of C4-2 cells. Gene microarray analyses revealed that AR knockdown under hormone-deprived conditions resulted in activation of genes involved in apoptosis, cell cycle regulation, protein synthesis, and tumorigenesis. To ensure that tumors were truly castration-resistant in vivo, inducible AR shRNA expressing C4-2 tumors were grown in castrated mice to an average volume of 450 mm3. In all of the animals, serum PSA decreased, and in 50% of them, there was complete tumor regression and disappearance of serum PSA. Conclusions: Whereas castration is ineffective in castration-resistant prostate tumors, knockdown of AR can decrease serum PSA, inhibit tumor growth, and frequently cause tumor regression. This study is the first direct evidence that knockdown of AR is a viable therapeutic strategy for treatment of prostate tumors that have already progressed to the castration-resistant state.

Ecological and social characterization of key resource areas in Kenyan rangelands

Key resource areas (KRAs), defined as dry season foraging zones for herbivores, were studied relative to the more extensive outlying rangeland areas (non-KRAs) in Kenya. Field surveys with pastoralists, ranchers, scientists and government officials delineated KRAs on the ground. Identified KRAs were mapped based on global positioning and local experts' information on KRAs accessibility and ecological attributes. Using the map of known KRAs and non-KRAs, we examined characteristics of soils, climate, topography, land use/cover attributes at KRAs relative to non-KRAs. How and why do some areas (KRAs) support herbivores during droughts when forage is scarce in other areas of the landscape? We hypothesized that KRAs have fundamental ecological and socially determined attributes that enable them to provide forage during critical times and we sought to characterize some of those attributes in this study. At the landscape level, KRAs took different forms based on forage availability during the dry season but generally occurred in locations of the landscape with aseasonal water availability and/or difficult to access areas during wet season forage abundance. Greenness trends for KRAs versus non-KRAs were evaluated with a 22-year dataset of Normalized Difference Vegetation Index (NDVI). Field surveys of KRAs provided qualitative information on KRAs as dry season foraging zones. At the scale of the study, soil attributes did not significantly differ for KRAs compared to non-KRAs. Slopes of KRA were generally steeper compared to non-KRAs and elevation was higher at KRAs. Field survey respondents indicated that animals and humans generally avoid difficult to access hilly areas using them only when all other easily accessible rangeland is depleted of forage during droughts. Understanding the nature of KRAs will support identification, protection and restoration of critical forage hotspots for herbivores by strengthening rangeland inventory, monitoring, policy formulation, and conservation efforts to improve habitats and human welfare. (c) 2007 Elsevier Ltd. All rights reserved.

Influences of age and mechanical stability on volume, microstructure, and mineralization of the fracture callus during bone healing : Is osteoclast activity the key to age-related impaired healing?

Earlier studies have shown that the influence of fixation stability on bone healing diminishes with advanced age. The goal of this study was to unravel the relationship between mechanical stimulus and age on callus competence at a tissue level. Using 3D in vitro micro-computed tomography derived metrics, 2D in vivo radiography, and histology, we investigated the influences of age and varying fixation stability on callus size, geometry, microstructure, composition, remodeling, and vascularity. Compared were four groups with a 1.5-mm osteotomy gap in the femora of Sprague–Dawley rats: Young rigid (YR), Young semirigid (YSR), Old rigid (OR), Old semirigid (OSR). Hypothesis was that calcified callus microstructure and composition is impaired due to the influence of advanced age, and these individuals would show a reduced response to fixation stabilities. Semirigid fixations resulted in a larger ΔCSA (Callus cross-sectional area) compared to rigid groups. In vitro μCT analysis at 6 weeks postmortem showed callus bridging scores in younger animals to be superior than their older counterparts (pb0.01). Younger animals showed (i) larger callus strut thickness (pb0.001), (ii) lower perforation in struts (pb0.01), and (iii) higher mineralization of callus struts (pb0.001). Callus mineralization was reduced in young animals with semirigid fracture fixation but remained unaffected in the aged group. While stability had an influence, age showed none on callus size and geometry of callus. With no differences observed in relative osteoid areas in the callus ROI, old as well as semirigid fixated animals showed a higher osteoclast count (pb0.05). Blood vessel density was reduced in animals with semirigid fixation (pb0.05). In conclusion, in vivo monitoring indicated delayed callus maturation in aged individuals. Callus bridging and callus competence (microstructure and mineralization) were impaired in individuals with an advanced age. This matched with increased bone resorption due to higher osteoclast numbers. Varying fixator configurations in older individuals did not alter the dominant effect of advanced age on callus tissue mineralization, unlike in their younger counterparts. Age-associated influences appeared independent from stability. This study illustrates the dominating role of osteoclastic activity in age-related impaired healing, while demonstrating the optimization of fixation parameters such as stiffness appeared to be less effective in influencing healing in aged individuals.