The phylogeny of Ureaplasma urealyticum based on the mba gene fragment

Sequencing of mba gene fragments of reference strains of Ureaplasma urealyticum serovars 1, 3, 6, 14, in addition to 33 clinical U. urealyticum isolates is reported. A phylogenetic tree deduced from an alignment of these sequences clearly demonstrates two major clusters (confidence limit 100%), which equate to the parvo and T960 biovars, and five types which we have designated mba 1, 3, 6, 8 and X. These relationships are supported by bootstrap analysis. Polymorphisms within the mba fragment of types mba 1, 3, and 6 were used to define nine subtypes (mba 1a, 1b, 3a, 3b, 3c, 3d, 3e, 6a, and 6b) thus facilitating high resolution typing of U. urealyticum. Inclusion of the reference strains for serovars 1, 3, 6, and 8 in the mba typing scheme showed that the results of this analysis are broadly consistent with currently accepted serotyping. In addition a ure gene fragment from nine of the clinical isolates was amplified and sequenced. Comparisons of the sequences clearly distinguished the two biovars of U. urealyticum; however this fragment was invariant within the parvo biovar. This study has shown that the sequence of the mba can reveal the fine details of the relationships between U. urealyticum isolates and also supports the significant evolutionary gap between the two biovars.

Chlamydia pneumoniae : modern insights into an ancient pathogen

Chlamydia pneumoniae is an enigmatic human and animal pathogen. Originally discovered in association with acute human respiratory disease, it is now associated with a remarkably wide range of chronic diseases as well as having a cosmopolitan distribution within the animal kingdom. Molecular typing studies suggest that animal strains are ancestral to human strains and that C. pneumoniae crossed from animals to humans as the result of at least one relatively recent zoonotic event. Whole genome analyses appear to support this concept – the human strains are highly conserved whereas the single animal strain that has been fully sequenced has a larger genome with several notable differences. When compared to the other, better known chlamydial species that is implicated in human infection, Chlamydia trachomatis, C. pneumoniae demonstrates pertinent differences in its cell biology, development, and genome structure. Here, we examine the characteristic facets of C. pneumoniae biology, offering insights into the diversity and evolution of this silent and ancient pathogen.

Comparison of antigen detection and quantitative PCR in the detection of chlamydial infection in koalas (Phascolarctos cinereus)

The gold standard method for detecting chlamydial infection in domestic and wild animals is PCR, but the technique is not suited to testing animals in the field when a rapid diagnosis is frequently required. The objective of this study was to compare the results of a commercially available enzyme immunoassay test for Chlamydia against a quantitative Chlamydia pecorum-specific PCR performed on swabs collected from the conjunctival sac, nasal cavity and urogenital sinuses of naturally infected koalas (Phascolarctos cinereus). The level of agreement for positive results between the two assays was low (43.2%). The immunoassay detection cut-off was determined as approximately 400 C. pecorum copies, indicating that the test was sufficiently sensitive to be used for the rapid diagnosis of active chlamydial infections.

Antigenic specificity of a monovalent versus polyvalent MOMP based Chlamydia pecorum vaccine in koalas (Phascolarctos cinereus)

Chlamydia continues to be a major pathogen of koalas. The bacterium is associated with ocular, respiratory and urogenital tract infections and a vaccine is considered the best option to limit the decline of mainland koala populations. Over the last 20 years, efforts to develop a chlamydial vaccine in humans have focussed on the use of the chlamydial major outer membrane protein (MOMP). Potential problems with the use of MOMP-based vaccines relate to the wide range of genetic diversity in its four variable domains. In the present study, we evaluated the immune response of koalas vaccinated with a MOMP-based C. pecorum vaccine formulated with genetically and serologically diverse MOMPs. Animals immunised with individual MOMPs developed strong antibody and lymphocyte proliferation responses to both homologous as well as heterologous MOMP proteins. Importantly, we also showed that vaccine induced antibodies which effectively neutralised various heterologous strains of koala C. pecorum in an in vitro assay. Finally, we also demonstrated that the immune responses in monovalent as well as polyvalent MOMP vaccine groups were able to recognise whole chlamydial elementary bodies, illustrating the feasibility of developing an effective MOMP based C. pecorum vaccine that could protect against a range of strains.

Characterization of in vitro Chlamydia muridarum persistence and utilization in an in vivo mouse model of Chlamydia vaccine

Problem: Chlamydia trachomatis genital tract infections are easily treated with antibiotics, however the majority of infections are asymptomatic and therefore untreated, highlighting the need for a vaccine. Because most infections are asymptomatic, vaccination could potentially be administered to individuals who may have an acute infection at that time. In such individuals the effect of vaccination on the existing infection is unknown; however one potential outcome could be the development of a persistent infection. In vitro chlamydial persistence has been well characterized in various strains, however there have been no reported studies in C. muridarum. Method of Study: We performed ultrastructural characterization, and transcriptome analysis of selected genes. We then used the transcriptional profiles of the selected genes to examine whether intranasal immunization of mice during an active genital infection would induce persistence in the upper reproductive tract of female mice. Results and Conclusions: We found that persistence developed in the oviducts of mice as a result of immunization. This is a significant finding, not only because it is the first time that C. muridarum persistence has been characterized in vitro, but also due to the fact that there is minimal characterization of in vivo persistence of any chlamydial species. This highlights the importance of the timing of vaccination in individuals.

Measurement of anisotropies of the kinetic energy and anharmonicity in pyrolytic graphite by neutron Compton scattering

Neutron Compton scattering (NCS) measurements of the anisotropy of the momentum distribution and the mean Laplacian of the interatomic potential ∇2V have been performed using electron volt neutrons, with wave vector transfers between 24 Å−1 and 98 Å−1. The measured momentum distribution of the atoms displays significantly more anisotropy than a calculation using a model density of states. We have observed anisotropies in ∇2V for the first time. The results suggest that the atomic potential is harmonic within the graphite planes, but anharmonic for vibrations perpendicular to the planes.

The temperature dependence of the momentum distribution of beryllium measured by neutron Compton scattering

In this paper we report a new neutron Compton scattering (NCS) measurement of the ground state single atom kinetic energy of polycrystalline beryllium at momentum transfers in the range 27}104 As ~1 and temperatures in the range 110}1150 K. The measurements have been made with the electron Volt spectrometer (eVS) at the ISIS facility and the measured kinetic energies are shown to be &10% higher than calculations made in the harmonic approximation.

Final-state effects in neutron Compton scattering measurements on zirconium deuteride and beryllium

We report inelastic neutron scattering measurements of the neutron Compton profile, J(y), for Be and for D in polycrystalline ZrD2 over a range of momentum transfers, q between 27 and 178 °A−1. The measurements were performed using the inverse geometry spectrometer eVS which is situated at the UK pulsed spallation neutron source ISIS. We have investigated deviations from impulse approximation (IA) scattering which are generically referred to as final state effects (FSEs) using a method described by Sears. This method allows both the magnitude and the q dependence of the FSE to be studied. Analysis of the measured data was compared with analysis of numerical simulations based on the harmonic approximation and good agreement was found for both ZrD2 and Be. Finally we have shown how (∇2V), where V is the interatomic potential, can be extracted from the antisymmetric component of J(y).

Development status and future prospects for a vaccine against Chlamydia trachomatis infection

Chlamydia trachomatis continues to be the most commonly reported sexually transmitted bacterial infection in many countries with more than 100 million new cases estimated annually. These acute infections translate into significant downstream health care costs, particularly for women, where complications can include pelvic inflammatory disease and other disease sequelae such as tubal factor infertility. Despite years of research, the immunological mechanisms responsible for protective immunity versus immunopathology are still not well understood, although it is widely accepted that T cell driven IFN-g and Th17 responses are critical for clearing infection. While antibodies are able to neutralize infections in vitro, alone they are not protective, indicating that any successful vaccine will need to elicit both arms of the immune response. In recent years, there has been an expansion in the number and types of antigens that have been evaluated as vaccines, and combined with the new array of mucosal adjuvants, this aspect of chlamydial vaccinology is showing promise. Most recently, the opportunities to develop successful vaccines have been given a significant boost with the development of a genetic transformation system for Chlamydia, as well as the identification of the key role of the chlamydial plasmid in virulence. While still remaining a major challenge, the development of a successful C.trachomatis vaccine is starting to look more likely.

Characterisation of enterovirus 71 replication kinetics in human colorectal cell line, HT29

Hand, Foot and Mouth Disease (HFMD), a contagious viral disease that commonly affects infants and children with blisters and flu like symptoms, is caused by a group of enteroviruses such as Enterovirus 71 (EV71) and coxsackievirus A16 (CA16). However some HFMD caused by EV71 may further develop into severe neurological complications such as encephalitis and meningitis. The route of transmission was postulated that the virus transmit from one person to another through direct contact of vesicular fluid or droplet from the infected or via faecal-oral route. To this end, this study utilised a human colorectal adenocarcinoma cell line (HT29) with epithelioid morphology as an in vitro model for the investigation of EV71 replication kinetics. Using qPCR, viral RNA was first detected in HT29 cells as early as 12 h post infection (hpi) while viral protein was first detected at 48 hpi. A significant change in HT29 cells’ morphology was also observed after 48 hpi. Furthermore HT29 cell viability also significantly decreased at 72 hpi. Together, data from this study demonstrated that co-culture of HT29 with EV71 is a useful in vitro model to study the pathogenesis of EV71

Molecular characterisation and expression analysis of interferon gamma in response to natural chlamydia infection in the koala, phascolarctos cinereus

Interferon gamma (IFNγ) is a key Th1 cytokine, with a principal role in the immune response against intracellular organisms such as Chlamydia. Along with being responsible for significant morbidity in human populations, Chlamydia is also responsible for wide spread infection and disease in many animal hosts, with reports that many Australian koala subpopulations are endemically infected. An understanding of the role played by IFNγ in koala chlamydial diseases is important for the establishment of better prophylactic and therapeutic approaches against chlamydial infection in this host. A limited number of IFNγ sequences have been published from marsupials and no immune reagents to measure expression have been developed. Through preliminary analysis of the koala transcriptome, we have identified the full coding sequence of the koala IFNγ gene. Transcripts were identified in spleen and lymph node tissue samples. Phylogenetic analysis demonstrated that koala IFNγ is closely related to other marsupial IFNγ sequences and more distantly related to eutherian mammals. To begin to characterise the role of this important cytokine in the koala's response to chlamydial infection, we developed a quantitative real time PCR assay and applied it to a small cohort of koalas with and without active chlamydial disease, revealing significant differences in expression patterns between the groups. Description of the IFNγ sequence from the koala will not only assist in understanding this species' response to its most important pathogen but will also provide further insight into the evolution of the marsupial immune system

The duration of Chlamydia muridarum genital tract infection and associated chronic pathological changes are reduced in IL-17 knockout mice but protection is not increased further by immunization

IL-17 is believed to be important for protection against extracellular pathogens, where clearance is dependent on neutrophil recruitment and local activation of epithelial cell defences. However, the role of IL-17 in protection against intracellular pathogens such as Chlamydia is less clear. We have compared (i) the course of natural genital tract C. muridarum infection, (ii) the development of oviduct pathology and (iii) the development of vaccine-induced immunity against infection in wild type (WT) BALB/c and IL-17 knockout mice (IL-17-/-) to determine if IL-17-mediated immunity is implicated in the development of infection-induced pathology and/or protection. Both the magnitude and duration of genital infection was significantly reduced in IL-17-/- mice compared to BALB/c. Similarly, hydrosalpinx was also greatly reduced in IL-17-/- mice and this correlated with reduced neutrophil and macrophage infiltration of oviduct tissues. Matrix metalloproteinase (MMP) 9 and MMP2 were increased in WT oviducts compared to IL-17-/- animals at day 7 post-infection. In contrast, oviducts from IL-17-/- mice contained higher MMP9 and MMP2 at day 21. Infection also elicited higher levels of Chlamydia-neutralizing antibody in serum of IL-17-/- mice than WT mice. Following intranasal immunization with C. muridarum Major Outer Membrane Protein (MOMP) and cholera toxin plus CpG adjuvants, significantly higher levels of chlamydial MOMP-specific IgG and IgA were found in serum and vaginal washes of IL-17-/- mice. T cell proliferation and IFNγ production by splenocytes was greater in WT animals following in vitro re-stimulation, however vaccination was only effective at reducing infection in WT, not IL-17-/- mice. Intranasal or transcutaneous immunization protected WT but not IL-17-/- mice against hydrosalpinx development. Our data show that in the absence of IL-17, the severity of C. muridarum genital infection and associated oviduct pathology are significantly attenuated, however neither infection or pathology can be reduced further by vaccination protocols that effectively protect WT mice.

A cohort study of Chlamydia trachomatis treatment failure in women : a study protocol

Background Chlamydia trachomatis is the most commonly diagnosed bacterial sexually transmitted infection in the developed world and diagnosis rates have increased dramatically over the last decade. Repeat infections of chlamydia are very common and may represent re-infection from an untreated partner or treatment failure. The aim of this cohort study is to estimate the proportion of women infected with chlamydia who experience treatment failure after treatment with 1 gram azithromycin. Methods/design This cohort study will follow women diagnosed with chlamydia for up to 56 days post treatment. Women will provide weekly genital specimens for further assay. The primary outcome is the proportion of women who are classified as having treatment failure 28, 42 or 56 days after recruitment. Comprehensive sexual behavior data collection and the detection of Y chromosome DNA and high discriminatory chlamydial genotyping will be used to differentiate between chlamydia re-infection and treatment failure. Azithromycin levels in high-vaginal specimens will be measured using a validated liquid chromatography – tandem mass spectrometry method to assess whether poor azithromycin absorption could be a cause of treatment failure. Chlamydia culture and minimal inhibitory concentrations will be performed to further characterize the chlamydia infections. Discussion Distinguishing between treatment failure and re-infection is important in order to refine treatment recommendations and focus infection control mechanisms. If a large proportion of repeat chlamydia infections are due to antibiotic treatment failure, then international recommendations on chlamydia treatment may need to be re-evaluated. If most are re-infections, then strategies to expedite partner treatment are necessary.

Identification of a serine protease inhibitor which causes inclusion vacuole reduction and is lethal to Chlamydia trachomatis

The mechanistic details of the pathogenesis of Chlamydia, an obligate intracellular pathogen of global importance, have eluded scientists due to the scarcity of traditional molecular genetic tools to investigate this organism. Here we report a chemical biology strategy that has uncovered the first essential protease for this organism. Identification and application of a unique CtHtrA inhibitor (JO146) to cultures of Chlamydia resulted in a complete loss of viable elementary body formation. JO146 treatment during the replicative phase of development resulted in a loss of Chlamydia cell morphology, diminishing inclusion size, and ultimate loss of inclusions from the host cells. This completely prevented the formation of viable Chlamydia elementary bodies. In addition to its effect on the human C. trachomatis strain, JO146 inhibited the viability of the mouse strain, Chlamydia muridarum, both in vitro and in vivo. Thus, we report a chemical biology approach to establish an essential role for Chlamydia CtHtrA. The function of CtHtrA for Chlamydia appears to be essential for maintenance of cell morphology during replicative the phase and these findings provide proof of concept that proteases can be targetted for anti-microbial therapy for intracellular pathogens.

Proof of concept : A bioinformatic and serological screening method for identifying new peptide antigens for Chlamydia trachomatis related sequelae in women

This study aimed to identify new peptide antigens from Chlamydia (C.) trachomatis in a proof of concept approach which could be used to develop an epitope-based serological diagnostic for C. trachomatis related infertility in women. A bioinformatics analysis was conducted examining several immunodominant proteins from C. trachomatis to identify predicted immunoglobulin epitopes unique to C. trachomatis. A peptide array of these epitopes was screened against participant sera. The participants (all female) were categorized into the following cohorts based on their infection and gynecological history; acute (single treated infection with C. trachomatis), multiple (more than one C. trachomatis infection, all treated), sequelae (PID or tubal infertility with a history of C. trachomatis infection), and infertile (no history of C. trachomatis infection and no detected tubal damage). The bioinformatics strategy identified several promising epitopes. Participants who reacted positively in the peptide 11 ELISA were found to have an increased likelihood of being in the sequelae cohort compared to the infertile cohort with an odds ratio of 16.3 (95% c.i. 1.65 – 160), with 95% specificity and 46% sensitivity (0.19-0.74). The peptide 11 ELISA has the potential to be further developed as a screening tool for use during the early IVF work up and provides proof of concept that there may be further peptide antigens which could be identified using bioinformatics and screening approaches.