Diversity of community acquired MRSA carrying the PVL gene in Queensland and New South Wales, Australia

Emergence and dissemination of community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) strains are being reported with increasing frequency in Australia and worldwide. These strains of CA-MRSA are genetically diverse and distinct in Australia. Genotyping of CA-MRSA using eight highly-discriminatory single nucleotide polymorphisms (SNPs) is a rapid and robust method for monitoring the dissemination of these strains in the community. In this study, a SNP genotyping method was used to investigate the molecular epidemiology of 249 community acquired non-multiresistant MRSA (nm-MRSA) isolates over a 12-month period from routine diagnostic specimens. A real-time PCR for the presence of Panton-Valentine leukocidin (PVL) was also performed on these isolates. The CA-MRSA isolates were sourced from a large private laboratory in Brisbane, Australia that serves a wide geographic region encompassing Queensland and Northern New South Wales. This study identified 16 different STs and 98% of the CA-MRSA isolates were positive for the PVL gene. The most common ST was ST93 with 41% of isolates testing positive for this clone.

Depth-dependent biomechanical and biochemical properties of fetal, newborn, and tissue-engineered articular cartilage

Adult articular cartilage has depth-dependent mechanical and biochemical properties which contribute to zone-specific functions. The compressive moduli of immature cartilage and tissue-engineered cartilage are known to be lower than those of adult cartilage. The objective of this study was to determine if such tissues exhibit depth-dependent compressive properties, and how these depth-varying properties were correlated with cell and matrix composition of the tissue. The compressive moduli of fetal and newborn bovine articular cartilage increased with depth (p < 0.05) by a factor of 4-5 from the top 0.1 mm (28 +/- 13 kPa, 141 +/- 10 kPa, respectively) to 1 mm deep into the tissue. Likewise, the glycosaminoglycan and collagen content increased with depth (both p < 0.001), and correlated with the modulus (both p < 0.01). In contrast, tissue-engineered cartilage formed by either layering or mixing cells from the superficial and middle zone of articular cartilage exhibited similarly soft regions at both construct surfaces, as exemplified by large equilibrium strains. The properties of immature cartilage may provide a template for developing tissue-engineered cartilage which aims to repair cartilage defects by recapitulating the natural development and growth processes. These results suggest that while depth-dependent properties may be important to engineer into cartilage constructs, issues other than cell heterogeneity must be addressed to generate such tissues. (c) 2005 Elsevier Ltd. All rights reserved.

Phylogeographic reconstruction of a bacterial species with high levels of lateral gene transfer

Background Phylogeographic reconstruction of some bacterial populations is hindered by low diversity coupled with high levels of lateral gene transfer. A comparison of recombination levels and diversity at seven housekeeping genes for eleven bacterial species, most of which are commonly cited as having high levels of lateral gene transfer shows that the relative contributions of homologous recombination versus mutation for Burkholderia pseudomallei is over two times higher than for Streptococcus pneumoniae and is thus the highest value yet reported in bacteria. Despite the potential for homologous recombination to increase diversity, B. pseudomallei exhibits a relative lack of diversity at these loci. In these situations, whole genome genotyping of orthologous shared single nucleotide polymorphism loci, discovered using next generation sequencing technologies, can provide very large data sets capable of estimating core phylogenetic relationships. We compared and searched 43 whole genome sequences of B. pseudomallei and its closest relatives for single nucleotide polymorphisms in orthologous shared regions to use in phylogenetic reconstruction. Results Bayesian phylogenetic analyses of >14,000 single nucleotide polymorphisms yielded completely resolved trees for these 43 strains with high levels of statistical support. These results enable a better understanding of a separate analysis of population differentiation among >1,700 B. pseudomallei isolates as defined by sequence data from seven housekeeping genes. We analyzed this larger data set for population structure and allele sharing that can be attributed to lateral gene transfer. Our results suggest that despite an almost panmictic population, we can detect two distinct populations of B. pseudomallei that conform to biogeographic patterns found in many plant and animal species. That is, separation along Wallace's Line, a biogeographic boundary between Southeast Asia and Australia. Conclusion We describe an Australian origin for B. pseudomallei, characterized by a single introduction event into Southeast Asia during a recent glacial period, and variable levels of lateral gene transfer within populations. These patterns provide insights into mechanisms of genetic diversification in B. pseudomallei and its closest relatives, and provide a framework for integrating the traditionally separate fields of population genetics and phylogenetics for other bacterial species with high levels of lateral gene transfer.

Effect of modular neck variation on bone and cement mantle mechanics around a total hip arthroplasty stem

Background Total hip arthroplasty carried out using cemented modular-neck implants provides the surgeon with greater intra-operative flexibility and allows more controlled stem positioning. Methods In this study, finite element models of a whole femur implanted with either the Exeter or with a new cemented modular-neck total hip arthroplasty (separate, neck and stem components) were developed. The changes in bone and cement mantle stress/strain were assessed for varying amounts of neck offset and version angle for the modular-neck device for two simulated physiological load cases: walking and stair climbing. Since the Exeter is the gold standard for polished cemented total hip arthroplasty stem design, bone and cement mantle stresses/strains in the modular-neck finite element models were compared with finite element results for the Exeter. Findings For the two physiological load cases, stresses and strains in the bone and cement mantle were similar for all modular-neck geometries. These results were comparable to the bone and cement mechanics surrounding the Exeter. These findings suggest that the Exeter and the modular neck device distribute stress to the surrounding bone and cement in a similar manner. Interpretation It is anticipated that the modular-neck device will have a similar short-term clinical performance to that of the Exeter, with the additional advantages of increased modularity.

Performance of square and inclined insulated rail joints based on field strain measurements

Insulated rail joints (IRJs) possess lower bending stiffness across the gap containing insulating endpost and hence are subjected to wheel impact. IRJs are either square cut or inclined cut to the longitudinal axis of the rails in a vertical plane. It is generally claimed that the inclined cut IRJs outperformed the square cut IRJs; however, there is a paucity of literature with regard to the relative structural merits of these two designs. This article presents comparative studies of the structural response of these two IRJs to the passage of wheels based on continuously acquired field data from joints strain-gauged closer to the source of impact. Strain signatures are presented in time, frequency, and avelet domains and the peak vertical and shear strains are systematically employed to examine the relative structural merits of the two IRJs subjected to similar real-life loading. It is shown that the inclined IRJs resist the wheel load with higher peak shear strains and lower peak vertical strains than that of the square IRJs.

An experimental research investigation into disused flat rail wagons as bridges with applications for low volume rural roads in Australia

An experimental laboratory investigation was carried out to assess the structural adequacy of a disused PHO Class Flat Bottom Rail Wagon (FRW) for a single lane low volume road bridge application as per the design provisions of the Australian Bridge Design Standard AS 5100(2004). The investigation also encompassed a review into the risk associated with the pre-existing damage in wagons incurred during their service life on rail. The main objective of the laboratory testing of the FRW was to physically measure its performance under the same applied traffic loading it would be required to resist as a road bridge deck. In order to achieve this a full width (5.2m) single lane, single span (approximately 10m), simply supported bridge would be required to be constructed and tested in a structural laboratory. However, the available clear spacing between the columns of the loading portal frame encountered within the laboratory was insufficient to accommodate the 5.2m wide bridge deck excluding clearance normally considered necessary in structural testing. Therefore, only half of the full scale bridge deck (single FRW of width 2.6m) was able to be accommodated and tested; with the continuity of the bridge deck in the lateral direction applied as boundary constraints along the full length of the FRW at six selected locations. This represents a novel approach not yet reported in the literature for bridge deck testing to the best of the knowledge of the author. The test was carried out under two loadings provided in AS 5100 (2004) – one stationary W80 wheel load and the second a moving axle load M1600. As the bridge investigated in the study is a single lane single span low volume road bridge, the risk of pre-existing damage and the expected high cycle fatigue failure potential was assessed as being minimal and hence the bridge deck was not tested structurally for fatigue/ fracture. The high axle load requirements have instead been focussed upon the investigation into the serviceability and ultimate limit state requirements. The testing regime adopted however involved extensive recording of strains and deflections at several critical locations of the FRW. Three locations of W80 point load and two locations of the M1600 Axle load were considered for the serviceability testing; the FRW was also tested under the ultimate load dictated by the M1600. The outcomes of the experimental investigation have demonstrated that the FRW is structurally adequate to resist the prescribed traffic loadings outlaid in AS 5100 (2004). As the loading was directly applied on to the FRW, the laboratory testing is assessed as being significantly conservative. The FRW bridge deck in the field would only resist the load transferred by the running platform, where, depending on the design, composite action might exist – thereby the share of the loading which needs to be resisted by the FRW would be smaller than the system tested in the lab. On this basis, a demonstration bridge is under construction at the time of writing this thesis and future research will involve field testing in order to assess its performance.

Detection of bacteriophage and respiratory viruses in droplets

Influenza is a widespread disease occurring in seasonal epidemics, and each year is responsible for up to 500,000 deaths worldwide. Influenza can develop into strains which cause severe symptoms and high mortality rates, and could potentially reach pandemic status if the virus’ properties allow easy transmission. Influenza is transmissible via contact with the virus, either directly (infected people) or indirectly (contaminated objects); via reception of large droplets over short distances (one metre or less); or through inhalation of aerosols containing the virus expelled by infected individuals during respiratory activities, that can remain suspended in the air and travel distances of more than one metre (the aerosol route). Aerosol transmission of viruses involves three stages: production of the droplets containing viruses; transport of the droplets and ability of a virus to remain intact and infectious; and reception of the droplets (via inhalation). Our understanding of the transmission of influenza viruses via the aerosol route is poor, and thus our ability to prevent a widespread outbreak is limited. This study explored the fate of viruses in droplets by investigating the effects of some physical factors on the recovery of both a bacteriophage model and influenza virus. Experiments simulating respiratory droplets were carried out using different types of droplets, generated from a commonly used water-like matrix, and also from an ‘artificial mucous’ matrix which was used to more closely resemble respiratory fluids. To detect viruses in droplets, we used the traditional plaque assay techniques, and also a sensitive, quantitative PCR assay specifically developed for this study. Our results showed that the artificial mucous suspension enhanced the recovery of infectious bacteriophage. We were able to report detection limits of infectious bacteriophage (no bacteriophage was detected by the plaque assay when aerosolised from a suspension of 103 PFU/mL, for three of the four droplet types tested), and that bacteriophage could remain infectious in suspended droplets for up to 20 minutes. We also showed that the nested real-time PCR assay was able to detect the presence of bacteriophage RNA where the plaque assay could not detect any intact particles. Finally, when applying knowledge from the bacteriophage experiments, we reported the quantitative recoveries of influenza viruses in droplets, which were more consistent and stable than we had anticipated. Influenza viruses can be detected up to 20 minutes (after aerosolisation) in suspended aerosols and possibly beyond. It also was detectable from nebulising suspensions with relatively low concentrations of viruses.

Human-specific E. coli single nucleotide polymorphism (SNP) genotypes detected in a South-East Queensland waterway, Australia

The World Health Organization recommends that the majority of water monitoring laboratories in the world should test for E. coli daily since thermotolerant coliforms and E. coli are key indicators for risk assessment of recreational waters. Recently, we developed a new SNP method for typing E. coli strains, by which human-specific genotypes were identified. Here, we report the presence of these previously described specific SNP profiles in environmental water, sourced from the Coomera River, located on South East Queensland, Australia, over a period of two years. This study tested for the presence of human-specific E. coli to ascertain whether hydrologic and anthropogenic activity plays a key role in the pollution of the investigated watershed or whether the pollution is from other sources. We found six human-specific SNP profiles and one animal-specific SNP profile consistently across sampling sites and times. We have demonstrated that our SNP genotyping method is able to rapidly identify and characterise human- and animal-specific E. coli isolates in water sources.

Comparative study of coagulase negative Staphylococci (CoNS) from clinical isolates, skin and nasal sources

Staphylococci are important pathogenic bacteria responsible for a range of diseases in humans. The most frequently isolated microorganisms in a hospital microbiology laboratory are staphylococci. The general classification of staphylococci divides them into two major groups; Coagulase-positive staphylococci (e.g. Staphylococcus aureus) and Coagulase-negative staphylococci (e.g. Staphylococcus epidermidis). Coagulase-negative staphylococcal (CoNS) isolates include a variety of species and many different strains but are often dominated by the most important organism of this group, S. epidermidis. Currently, these organisms are regarded as important pathogenic organisms causing infections related to prosthetic materials and surgical wounds. A significant number of S. epidermidis isolates are also resistant to different antimicrobial agents. Virulence factors in CoNS are not very clearly established and not well documented. S. epidermidis is evolving as a resistant and powerful microbe related to nosocomial infections because it has different properties which independently, and in combination, make it a successful infectious agent, especially in the hospital environment. Such characteristics include biofilm formation, drug resistance and the evolution of genetic variables. The purpose of this project was to develop a novel SNP genotyping method to genotype S. epidermidis strains originating from hospital patients and healthy individuals. High-Resolution Melt Analysis was used to assign binary typing profiles to both clinical and commensal strains using a new bioinformatics approach. The presence of antibiotic resistance genes and biofilm coding genes were also interrogated in these isolates.

Determination of stress-strain characteristics of railhead steel using image analysis

True stress-strain curve of railhead steel is required to investigate the behaviour of railhead under wheel loading through elasto-plastic Finite Element (FE) analysis. To reduce the rate of wear, the railhead material is hardened through annealing and quenching. The Australian standard rail sections are not fully hardened and hence suffer from non-uniform distribution of the material property; usage of average properties in the FE modelling can potentially induce error in the predicted plastic strains. Coupons obtained at varying depths of the railhead were, therefore, tested under axial tension and the strains were measured using strain gauges as well as an image analysis technique, known as the Particle Image Velocimetry (PIV). The head hardened steel exhibit existence of three distinct zones of yield strength; the yield strength as the ratio of the average yield strength provided in the standard (σyr=780MPa) and the corresponding depth as the ratio of the head hardened zone along the axis of symmetry are as follows: (1.17 σyr, 20%), (1.06 σyr, 20%- 80%) and (0.71 σyr, > 80%). The stress-strain curves exhibit limited plastic zone with fracture occurring at strain less than 0.1.

Performance testing of road bridge deck containing flat rail wagons

A road bridge containing disused flatbed rail wagons as the primary deck superstructure was performance tested in a low volume, high axle load traffic road in Queensland, Australia; some key results are presented in this paper. A fully laden truck of total weight 28.88 % of the serviceability design load prescribed in the Australian bridge code was used; its wheel positions were accurately captured using a high speed camera and synchronised with the real‐time deflections and strains measured at the critical members of the flat rail wagons. The strains remained well below the yield and narrated the existence of composite action between the reinforced concrete slab pavement and the wagon deck. A three dimensional grillage model was developed and calibrated using the test data, which established the structural adequacy of the rail wagons and the positive contribution of the reinforced concrete slab pavement to resist high axle traffic loads on a single lane bridge in the low volume roads network.

The role of the multiple banded antigen of ureaplasma parvum in intra-amniotic infection : major virulence factor or decoy?

The multiple banded antigen (MBA) is a predicted virulence factor of Ureaplasma species. Antigenic variation of the MBA is a potential mechanism by which ureaplasmas avoid immune recognition and cause chronic infections of the upper genital tract of pregnant women. We tested whether the MBA is involved in the pathogenesis of intra-amniotic infection and chorioamnionitis by injecting virulent or avirulent-derived ureaplasma clones (expressing single MBA variants) into the amniotic fluid of pregnant sheep. At 55 days of gestation pregnant ewes (n = 20) received intra-amniotic injections of virulent-derived or avirulent-derived U. parvum serovar 6 strains (2×104 CFU), or 10B medium (n = 5). Amniotic fluid was collected every two weeks post-infection and fetal tissues were collected at the time of surgical delivery of the fetus (140 days of gestation). Whilst chronic colonisation was established in the amniotic fluid of animals infected with avirulent-derived and virulent-derived ureaplasmas, the severity of chorioamnionitis and fetal inflammation was not different between these groups (p>0.05). MBA size variants (32–170 kDa) were generated in vivo in amniotic fluid samples from both the avirulent and virulent groups, whereas in vitro antibody selection experiments led to the emergence of MBA-negative escape variants in both strains. Anti-ureaplasma IgG antibodies were detected in the maternal serum of animals from the avirulent (40%) and virulent (55%) groups, and these antibodies correlated with increased IL-1β, IL-6 and IL-8 expression in chorioamnion tissue (p<0.05). We demonstrate that ureaplasmas are capable of MBA phase variation in vitro; however, ureaplasmas undergo MBA size variation in vivo, to potentially prevent eradication by the immune response. Size variation of the MBA did not correlate with the severity of chorioamnionitis. Nonetheless, the correlation between a maternal humoral response and the expression of chorioamnion cytokines is a novel finding. This host response may be important in the pathogenesis of inflammation-mediated adverse pregnancy outcomes.

Sandwiched carbon nanotube film as strain sensor

Two types of carbon nanotube nanocomposite strain sensors were prepared by mixing carbon nanotubes with epoxy (nanocomposite sensor) and sandwiching a carbon nanotube film between two epoxy layers (sandwich sensor). The conductivity, response and sensitivity to static and dynamic mechanical strains in these sensors were investigated. The nanocomposite sensor with 2-3 wt.% carbon nanotube demonstrated high sensitivity to mechanical strain and environmental temperature, with gauge factors of 5-8. On the other hand, a linear relationship between conductivity and dynamic mechanical strain was observed in the sandwich sensor. The sandwich sensor was also not sensitive to temperature although its strain sensitivity (gauge factor of about 3) was lower as compared with the nanocomposite sensor. Both sensors have excellent response to static and dynamic strains, thereby having great potential for strain sensing applications.

A screen for modifiers of epigenetic reprogramming

Epigenetic modifiers are the proteins involved in establishing and maintaining the epigenome of an organism. They are particularly important for development. Changes in epigenetic modifiers have been shown be lethal, or cause diseases. Our laboratory has developed an ENU mutagenesis screen to produce mouse mutants displaying altered epigenetic gene silencing. The screen relies on a GFP transgene that is expressed in red blood cells in a variegated manner. In the orginal transgenic FVB mice expression occurs in approximately 55% of red blood cells. During the course of my Masters, I characterised four different Mommes (Modifiers of murine metastable epiallele), MommeD32, MommeD33, MommeD35 and MommeD36. For each Momme, I identified the underlying mutation, and observed the corresponding phenotype. In MommeD32 the causative mutation is in Dnmt1, (DNA methyltransferase 1). This gene was previously identified in the screen, as MommeD2, and the new allele, MommeD32 has a change in the BAH domain of the protein. MommeD33 is the result of a change at the transgene itself. MommeD35 carries a mutation in Suv39h1 (suppressor of variegation 3-9 homolog 1). This gene has not previously been identified in the screen, but it is a known epigenetic modifier. MommeD36 had the same ENU treated sire as MommeD32, and I found that it has the same mutation as MommeD32. These mutant strains provide valuable tools that can be used to further our knowledge of epigenetic reprogramming. An example being the cancer study done with MommeD9 which has a mutation in Trim28. By crossing MommeD9+/- mutant mice with Trp53+/- mice, it can be seen if Trim28 has an effect on the rate of tumour genesis. However no clear effect of Trim28 haploinsufficiency can be observed in Trp53+/- mice.

Ureaplasma parvum : understanding the complexities of intra-amniotic infection in an ovine model

The human Ureaplasma species are the most frequently isolated bacteria from the upper genital tract of pregnant women and can cause clinically asymptomatic, intra-uterine infections, which are difficult to treat with antimicrobials. Ureaplasma infection of the upper genital tract during pregnancy has been associated with numerous adverse outcomes including preterm birth, chorioamnionitis and neonatal respiratory diseases. The mechanisms by which ureaplasmas are able to chronically colonise the amniotic fluid and avoid eradication by (i) the host immune response and (ii) maternally-administered antimicrobials, remain virtually unexplored. To address this gap within the literature, this study investigated potential mechanisms by which ureaplasmas are able to cause chronic, intra-amniotic infections in an established ovine model. In this PhD program of research the effectiveness of standard, maternal erythromycin for the treatment of chronic, intra-amniotic ureaplasma infections was evaluated. At 55 days of gestation pregnant ewes received an intra-amniotic injection of either: a clinical Ureaplasma parvum serovar 3 isolate that was sensitive to macrolide antibiotics (n = 16); or 10B medium (n = 16). At 100 days of gestation, ewes were then randomised to receive either maternal erythromycin treatment (30 mg/kg/day for four days) or no treatment. Ureaplasmas were isolated from amniotic fluid, chorioamnion, umbilical cord and fetal lung specimens, which were collected at the time of preterm delivery of the fetus (125 days of gestation). Surprisingly, the numbers of ureaplasmas colonising the amniotic fluid and fetal tissues were not different between experimentally-infected animals that received erythromycin treatment or infected animals that did not receive treatment (p > 0.05), nor were there any differences in fetal inflammation and histological chorioamnionitis between these groups (p > 0.05). These data demonstrate the inability of maternal erythromycin to eradicate intra-uterine ureaplasma infections. Erythromycin was detected in the amniotic fluid of animals that received antimicrobial treatment (but not in those that did not receive treatment) by liquid chromatography-mass spectrometry; however, the concentrations were below therapeutic levels (<10 – 76 ng/mL). These findings indicate that the ineffectiveness of standard, maternal erythromycin treatment of intra-amniotic ureaplasma infections may be due to the poor placental transfer of this drug. Subsequently, the phenotypic and genotypic characteristics of ureaplasmas isolated from the amniotic fluid and chorioamnion of pregnant sheep after chronic, intra-amniotic infection and low-level exposure to erythromycin were investigated. At 55 days of gestation twelve pregnant ewes received an intra-amniotic injection of a clinical U. parvum serovar 3 isolate, which was sensitive to macrolide antibiotics. At 100 days of gestation, ewes received standard maternal erythromycin treatment (30 mg/kg/day for four days, n = 6) or saline (n = 6). Preterm fetuses were surgically delivered at 125 days of gestation and ureaplasmas were cultured from the amniotic fluid and the chorioamnion. The minimum inhibitory concentrations (MICs) of erythromycin, azithromycin and roxithromycin were determined for cultured ureaplasma isolates, and antimicrobial susceptibilities were different between ureaplasmas isolated from the amniotic fluid (MIC range = 0.08 – 1.0 mg/L) and chorioamnion (MIC range = 0.06 – 5.33 mg/L). However, the increased resistance to macrolide antibiotics observed in chorioamnion ureaplasma isolates occurred independently of exposure to erythromycin in vivo. Remarkably, domain V of the 23S ribosomal RNA gene (which is the target site of macrolide antimicrobials) of chorioamnion ureaplasmas demonstrated significant variability (125 polymorphisms out of 422 sequenced nucleotides, 29.6%) when compared to the amniotic fluid ureaplasma isolates and the inoculum strain. This sequence variability did not occur as a consequence of exposure to erythromycin, as the nucleotide substitutions were identical between chorioamnion ureaplasmas isolated from different animals, including those that did not receive erythromycin treatment. We propose that these mosaic-like 23S ribosomal RNA gene sequences may represent gene fragments transferred via horizontal gene transfer. The significant differences observed in (i) susceptibility to macrolide antimicrobials and (ii) 23S ribosomal RNA sequences of ureaplasmas isolated from the amniotic fluid and chorioamnion suggests that the anatomical site from which they were isolated may exert selective pressures that alter the socio-microbiological structure of the bacterial population, by selecting for genetic changes and altered antimicrobial susceptibility profiles. The final experiment for this PhD examined antigenic size variation of the multiple banded antigen (MBA, a surface-exposed lipoprotein and predicted ureaplasmal virulence factor) in chronic, intra-amniotic ureaplasma infections. Previously defined ‘virulent-derived’ and ‘avirulent-derived’ clonal U. parvum serovar 6 isolates (each expressing a single MBA protein) were injected into the amniotic fluid of pregnant ewes (n = 20) at 55 days of gestation, and amniotic fluid was collected by amniocentesis every two weeks until the time of near-term delivery of the fetus (at 140 days of gestation). Both the avirulent and virulent clonal ureaplasma strains generated MBA size variants (ranging in size from 32 – 170 kDa) within the amniotic fluid of pregnant ewes. The mean number of MBA size variants produced within the amniotic fluid was not different between the virulent (mean = 4.2 MBA variants) and avirulent (mean = 4.6 MBA variants) ureaplasma strains (p = 0.87). Intra-amniotic infection with the virulent strain was significantly associated with the presence of meconium-stained amniotic fluid (p = 0.01), which is an indicator of fetal distress in utero. However, the severity of histological chorioamnionitis was not different between the avirulent and virulent groups. We demonstrated that ureaplasmas were able to persist within the amniotic fluid of pregnant sheep for 85 days, despite the host mounting an innate and adaptive immune response. Pro-inflammatory cytokines (interleukin (IL)-1â, IL-6 and IL-8) were elevated within the chorioamnion tissue of pregnant sheep from both the avirulent and virulent treatment groups, and this was significantly associated with the production of anti-ureaplasma IgG antibodies within maternal sera (p < 0.05). These findings suggested that the inability of the host immune response to eradicate ureaplasmas from the amniotic cavity may be due to continual size variation of MBA surface-exposed epitopes. Taken together, these data confirm that ureaplasmas are able to cause long-term in utero infections in a sheep model, despite standard antimicrobial treatment and the development of a host immune response. The overall findings of this PhD project suggest that ureaplasmas are able to cause chronic, intra-amniotic infections due to (i) the limited placental transfer of erythromycin, which prevents the accumulation of therapeutic concentrations within the amniotic fluid; (ii) the ability of ureaplasmas to undergo rapid selection and genetic variation in vivo, resulting in ureaplasma isolates with variable MICs to macrolide antimicrobials colonising the amniotic fluid and chorioamnion; and (iii) antigenic size variation of the MBA, which may prevent eradication of ureaplasmas by the host immune response and account for differences in neonatal outcomes. The outcomes of this program of study have improved our understanding of the biology and pathogenesis of this highly adapted microorganism.