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

TLR2, but Not TLR4, is required for effective host defence against Chlamydia respiratory tract infection in early life

Chlamydia pneumoniae commonly causes respiratory tract infections in children, and epidemiological investigations strongly link infection to the pathogenesis of asthma. The immune system in early life is immature and may not respond appropriately to pathogens. Toll-like receptor (TLR)2 and 4 are regarded as the primary pattern recognition receptors that sense bacteria, however their contribution to innate and adaptive immunity in early life remains poorly defined. We investigated the role of TLR2 and 4 in the induction of immune responses to Chlamydia muridarum respiratory infection, in neonatal wild-type (Wt) or TLR2-deficient (−/−), 4−/− or 2/4−/− BALB/c mice. Wt mice had moderate disease and infection. TLR2−/− mice had more severe disease and more intense and prolonged infection compared to other groups. TLR4−/− mice were asymptomatic. TLR2/4−/− mice had severe early disease and persistent infection, which resolved thereafter consistent with the absence of symptoms in TLR4−/− mice. Wt mice mounted robust innate and adaptive responses with an influx of natural killer (NK) cells, neutrophils, myeloid (mDCs) and plasmacytoid (pDCs) dendritic cells, and activated CD4+ and CD8+ T-cells into the lungs. Wt mice also had effective production of interferon (IFN)γ in the lymph nodes and lung, and proliferation of lymph node T-cells. TLR2−/− mice had more intense and persistent innate (particularly neutrophil) and adaptive cell responses and IL-17 expression in the lung, however IFNγ responses and T-cell proliferation were reduced. TLR2/4−/− mice had reduced innate and adaptive responses. Most importantly, neutrophil phagocytosis was impaired in the absence of TLR2. Thus, TLR2 expression, particularly on neutrophils, is required for effective control of Chlamydia respiratory infection in early life. Loss of control of infection leads to enhanced but ineffective TLR4-mediated inflammatory responses that prolong disease symptoms. This indicates that TLR2 agonists may be beneficial in the treatment of early life Chlamydia infections and associated diseases.

Chlamydia muridarum lung infection in infants alters hematopoietic cells to promote allergic airway disease in mice

Background Viral and bacterial respiratory tract infections in early-life are linked to the development of allergic airway inflammation and asthma. However, the mechanisms involved are not well understood. We have previously shown that neonatal and infant, but not adult, chlamydial lung infections in mice permanently alter inflammatory phenotype and physiology to increase the severity of allergic airway disease by increasing lung interleukin (IL)-13 expression, mucus hyper-secretion and airway hyper-responsiveness. This occurred through different mechanisms with infection at different ages. Neonatal infection suppressed inflammatory responses but enhanced systemic dendritic cell:T-cell IL-13 release and induced permanent alterations in lung structure (i.e., increased the size of alveoli). Infant infection enhanced inflammatory responses but had no effect on lung structure. Here we investigated the role of hematopoietic cells in these processes using bone marrow chimera studies. Methodology/Principal Findings Neonatal (<24-hours-old), infant (3-weeks-old) and adult (6-weeks-old) mice were infected with C. muridarum. Nine weeks after infection bone marrow was collected and transferred into recipient age-matched irradiated naïve mice. Allergic airway disease was induced (8 weeks after adoptive transfer) by sensitization and challenge with ovalbumin. Reconstitution of irradiated naïve mice with bone marrow from mice infected as neonates resulted in the suppression of the hallmark features of allergic airway disease including mucus hyper-secretion and airway hyper-responsiveness, which was associated with decreased IL-13 levels in the lung. In stark contrast, reconstitution with bone marrow from mice infected as infants increased the severity of allergic airway disease by increasing T helper type-2 cell cytokine release (IL-5 and IL-13), mucus hyper-secretion, airway hyper-responsiveness and IL-13 levels in the lung. Reconstitution with bone marrow from infected adult mice had no effects. Conclusions These results suggest that an infant chlamydial lung infection results in long lasting alterations in hematopoietic cells that increases the severity of allergic airway disease in later-life.

Increased risk of hospitalization for acute lower respiratory tract infection among Australian Indigenous infants 5-23 months of age following pneumococcal vaccination : a cohort study

Background Australian Indigenous children are the only population worldwide to receive the 7-valent pneumococcal conjugate vaccine (7vPCV) at 2, 4, and 6 months of age and the 23-valent pneumococcal polysaccharide vaccine (23vPPV) at 18 months of age. We evaluated this program's effectiveness in reducing the risk of hospitalization for acute lower respiratory tract infection (ALRI) in Northern Territory (NT) Indigenous children aged 5-23 months. Methods We conducted a retrospective cohort study involving all NT Indigenous children born from 1 April 2000 through 31 October 2004. Person-time at-risk after 0, 1, 2, and 3 doses of 7vPCV and after 0 and 1 dose of 23vPPV and the number of ALRI following each dose were used to calculate dose-specific rates of ALRI for children 5-23 months of age. Rates were compared using Cox proportional hazards models, with the number of doses of each vaccine serving as time-dependent covariates. Results There were 5482 children and 8315 child-years at risk, with 2174 episodes of ALRI requiring hospitalization (overall incidence, 261 episodes per 1000 child-years at risk). Elevated risk of ALRI requiring hospitalization was observed after each dose of the 7vPCV vaccine, compared with that for children who received no doses, and an even greater elevation in risk was observed after each dose of the 23vPPV ( adjusted hazard ratio [HR] vs no dose, 1.39; 95% confidence interval [CI], 1.12-1.71;). Risk was highest among children Pp. 002 vaccinated with the 23vPPV who had received < 3 doses of the 7vPCV (adjusted HR, 1.81; 95% CI, 1.32-2.48). Conclusions Our results suggest an increased risk of ALRI requiring hospitalization after pneumococcal vaccination, particularly after receipt of the 23vPPV booster. The use of the 23vPPV booster should be reevaluated.

Risks for HIV infection among male street laborers in urban Vietnam

Risks for HIV infection remain unknown in male street laborers. This research investigates patterns of self-reported risk behaviors among these men in urban Vietnam. In a cross-sectional survey using a social mapping technique, 450 men, mostly low-skilled and unregistered migrant laborers across 13 districts in Hanoi were approached for interviews. The study revealed that male street laborers were at high risk of acquiring and transmitting HIV. One in every 12 men reported homosexual or bisexual behavior. These men on average had three sexual partners within the preceding year, and condom use was inconsistent. Close to 95 % of the men had reported sexual encounters with regular partners. One-third with commercial sex workers (CSW) and 24.2 % with casual partners, but just under one-third had ever used condoms with regular partners and CSWs and very few (17.6 %) with casual partners at their last sexual encounter. 17.11 % used illicit drugs sometimes, with 66.7 % of them frequently sharing injecting equipment with peers. These men had limited HIV knowledge; 51.4 % incorrectly believed that, once you trust your partner, you no longer need to use condoms and 42.4 % believed that you can tell by looking at someone if they have HIV. Access to HIV prevention was also limited; only 19.8 % of men had been tested for HIV during the previous 12 months, almost 10 % of whom neither returned for the result nor knew their HIV status. The study provides interesting directions for future research and suggests ways to effectively design prevention strategies for these men.

The IL-6 response to Chlamydia from primary reproductive epithelial cells is highly variable and may be involved in differential susceptibility to the immunopathological consequences of chlamydial infection

Background Chlamydia trachomatis infection results in reproductive damage in some women. The process and factors involved in this immunopathology are not well understood. This study aimed to investigate the role of primary human cellular responses to chlamydial stress response proteases and chlamydial infection to further identify the immune processes involved in serious disease sequelae. Results Laboratory cell cultures and primary human reproductive epithelial cultures produced IL-6 in response to chlamydial stress response proteases (CtHtrA and CtTsp), UV inactivated Chlamydia, and live Chlamydia. The magnitude of the IL-6 response varied considerably (up to 1000 pg ml-1) across different primary human reproductive cultures. Thus different levels of IL-6 production by reproductive epithelia may be a determinant in disease outcome. Interestingly, co-culture models with either THP-1 cells or autologous primary human PBMC generally resulted in increased levels of IL-6, except in the case of live Chlamydia where the level of IL-6 was decreased compared to the epithelial cell culture only, suggesting this pathway may be able to be modulated by live Chlamydia. PBMC responses to the stress response proteases (CtTsp and CtHtrA) did not significantly vary for the different participant cohorts. Therefore, these proteases may possess conserved innate PAMPs. MAP kinases appeared to be involved in this IL-6 induction from human cells. Finally, we also demonstrated that IL-6 was induced by these proteins and Chlamydia from mouse primary reproductive cell cultures (BALB/C mice) and mouse laboratory cell models. Conclusions We have demonstrated that IL-6 may be a key factor for the chlamydial disease outcome in humans, given that primary human reproductive epithelial cell culture showed considerable variation in IL-6 response to Chlamydia or chlamydial proteins, and that the presence of live Chlamydia (but not UV killed) during co-culture resulted in a reduced IL-6 response suggesting this response may be moderated by the presence of the organism.

Immunity against a Chlamydia infection and disease may be determined by a balance of IL-17 signaling

Most vaccines developed against Chlamydia using animal models provide partial protection against a genital tract infection. However, protection against the oviduct pathology associated with infertility is highly variable and often has no defining immunological correlate. When comparing two adjuvants (CTA1-DD and a combination of Cholera toxin plus CpG- oligodeoxynucleotide–CT/CpG) combined with the chlamydial major outer membrane protein (MOMP) antigen and delivered via the intranasal (IN), sublingual (SL) or transcutaneous (TC) routes, we identified two vaccine groups with contrasting outcomes following infection. SL immunization with MOMP/CTA1-DD induced a 70% reduction in the incidence of oviduct pathology, without significantly altering the course of infection. Conversely, IN immunization with MOMP/CT/CpG prevented an ascending infection, but not the oviduct pathology. This anomaly presented a unique opportunity to study the mechanisms by which vaccines can prevent oviduct pathology, other than by controlling the infection. The IL-17 signaling in the oviducts was found to associate with both the enhancement of immunity to infection and the development of oviduct pathology. This conflicting role of IL-17 may provide some explanation for the discordance in protection between infection and disease and suggests that controlling immunopathology, as opposed to the rapid eradication of the infection, may be essential for an effective human chlamydial vaccine that prevents infertility.

The mouse model of Chlamydia genital tract infection: a review of infection, disease, immunity and vaccine development

Chlamydia trachomatis is the most common sexually transmitted bacterial infection worldwide. The impact of this pathogen on human reproduction has intensified research efforts to better understand chlamydial infection and pathogenesis. Whilst there are animal models available that mimic the many aspects of human chlamydial infection, the mouse is regarded as the most practical and widely used of the models. Studies in mice have greatly contributed to our understanding of the host-pathogen interaction and provided an excellent medium for evaluating vaccines. Here we explore the advantages and disadvantages of all animal models of chlamydial genital tract infection, with a focus on the murine model and what we have learnt from it so far.

Preliminary characterisation of tumor necrosis factor alpha and interleukin-10 responses to chlamydia pecorum infection in the koala (Phascolarctos cinereus)

Debilitating infectious diseases caused by Chlamydia are major contributors to the decline of Australia's iconic native marsupial species, the koala (Phascolarctos cinereus). An understanding of koala chlamydial disease pathogenesis and the development of effective strategies to control infections continue to be hindered by an almost complete lack of species-specific immunological reagents. The cell-mediated immune response has been shown to play an influential role in the response to chlamydial infection in other hosts. The objective of this study, hence, was to provide preliminary data on the role of two key cytokines, pro-inflammatory tumour necrosis factor alpha (TNFα) and anti-inflammatory interleukin 10 (IL10), in the koala Chlamydia pecorum response. Utilising sequence homology between the cytokine sequences obtained from several recently sequenced marsupial genomes, this report describes the first mRNA sequences of any koala cytokine and the development of koala specific TNFα and IL10 real-time PCR assays to measure the expression of these genes from koala samples. In preliminary studies comparing wild koalas with overt chlamydial disease, previous evidence of C. pecorum infection or no signs of C. pecorum infection, we revealed strong but variable expression of TNFα and IL10 in wild koalas with current signs of chlamydiosis. The description of these assays and the preliminary data on the cell-mediated immune response of koalas to chlamydial infection paves the way for future studies characterising the koala immune response to a range of its pathogens while providing reagents to assist with measuring the efficacy of ongoing attempts to develop a koala chlamydial vaccine.

Vaccination of koalas with a recombinant Chlamydia pecorum major outer membrane protein induces antibodies of different specificity compared to those following a natural live infection

Chlamydial infection in koalas is common across the east coast of Australia and causes significant morbidity, infertility and mortality. An effective vaccine to prevent the adverse consequences of chlamydial infections in koalas (particularly blindness and infertility in females) would provide an important management tool to prevent further population decline of this species. An important step towards developing a vaccine in koalas is to understand the host immune response to chlamydial infection. In this study, we used the Pepscan methodology to identify B cell epitopes across the Major Outer Membrane Protein (MOMP) of four C. pecorum strains/genotypes that are recognized, either following (a) natural live infection or (b) administration of a recombinant MOMP vaccine. Plasma antibodies from the koalas naturally infected with a C. pecorum G genotype strain recognised the epitopes located in the variable domain (VD) four of MOMP G and also VD4 of MOMP H. By comparison, plasma antibodies from an animal infected with a C. pecorum F genotype strain recognised epitopes in VD1, 2 and 4 of MOMP F, but not from other genotype MOMPs. When Chlamydia-free koalas were immunised with recombinant MOMP protein they produced antibodies not only against epitopes in the VDs but also in conserved domains of MOMP. Naturally infected koalas immunised with recombinant MOMP protein also produced antibodies against epitopes in the conserved domains. This work paves the way for further refinement of a MOMP-based Chlamydia vaccine that will offer wide cross-protection against the variety of chlamydial infections circulating in wild koala populations.

Projecting the impact of climate change on the transmission of Ross River virus : methodological challenges and research needs

Ross River virus (RRV) is the most common vector-borne disease in Australia. It is vitally important to make appropriate projections on the future spread of RRV under various climate change scenarios because such information is essential for policy-makers to identify vulnerable communities and to better manage RRV epidemics. However, there are many methodological challenges in projecting the impact of climate change on the transmission of RRV disease. This study critically examined the methodological issues and proposed possible solutions. A literature search was conducted between January and October 2012, using the electronic databases Medline, Web of Science and PubMed. Nineteen relevant papers were identified. These studies demonstrate that key challenges for projecting future climate change on RRV disease include: (1) a complex ecology (e.g. many mosquito vectors, immunity, heterogeneous in both time and space); (2) unclear interactions between social and environmental factors; and (3) uncertainty in climate change modelling and socioeconomic development scenarios. Future risk assessments of climate change will ultimately need to better understand the ecology of RRV disease and to integrate climate change scenarios with local socioeconomic and environmental factors, in order to develop effective adaptation strategies to prevent or reduce RRV transmission.

Prevention of surgical site infection in total joint arthroplasty : an international tertiary care center survery

Background Prevention strategies are critical to reduce infection rates in total joint arthroplasty (TJA), but evidence-based consensus guidelines on prevention of surgical site infection (SSI) remain heterogeneous and do not necessarily represent this particular patient population. Questions/Purposes What infection prevention measures are recommended by consensus evidence-based guidelines for prevention of periprosthetic joint infection? How do these recommendations compare to expert consensus on infection prevention strategies from orthopedic surgeons from the largest international tertiary referral centers for TJA? Patients and Methods A review of consensus guidelines was undertaken as described by Merollini et al. Four clinical guidelines met inclusion criteria: Centers for Disease Control and Prevention's, British Orthopedic Association, National Institute of Clinical Excellence's, and National Health and Medical Research Council's (NHMRC). Twenty-eight recommendations from these guidelines were used to create an evidence-based survey of infection prevention strategies that was administered to 28 orthopedic surgeons from members of the International Society of Orthopedic Centers. The results between existing consensus guidelines and expert opinion were then compared. Results Recommended strategies in the guidelines such as prophylactic antibiotics, preoperative skin preparation of patients and staff, and sterile surgical attire were considered critically or significantly important by the surveyed surgeons. Additional strategies such as ultraclean air/laminar flow, antibiotic cement, wound irrigation, and preoperative blood glucose control were also considered highly important by surveyed surgeons, but were not recommended or not uniformly addressed in existing guidelines on SSI prevention. Conclusion Current evidence-based guidelines are incomplete and evidence should be updated specifically to address patient needs undergoing TJA.

Chlamydia pneumoniae infection of Dentritic cells and its role in asthma exacerbations

Chlamydia infections are associated with exacerbations of asthma however the mechanisms are poorly understood. In this thesis we infected dendritic cells from healthy controls and asthmatic patients to determine if the immune response to chlamydial infection by these key immune cells could explain this association of chlamydial infection with asthma attacks. Infected dendritic cells from asthmatic patients showed increased expression of multiple inflammatory cytokine genes and genes for several tissue remodelling proteins, suggesting that infected dendritic cells play a central role in driving the airways damage associated with asthma. The findings provide a greater understanding of the role of infections in asthma and may provide a basis for new therapies to treat this important disease.

Slaving and release in co-infection control

Background Animal and human infection with multiple parasite species is the norm rather than the exception, and empirical studies and animal models have provided evidence for a diverse range of interactions among parasites. We demonstrate how an optimal control strategy should be tailored to the pathogen community and tempered by species-level knowledge of drug sensitivity with use of a simple epidemiological model of gastro-intestinal nematodes. Methods We construct a fully mechanistic model of macroparasite co-infection and use it to explore a range of control scenarios involving chemotherapy as well as improvements to sanitation. Results Scenarios are presented whereby control not only releases a more resistant parasite from antagonistic interactions, but risks increasing co-infection rates, exacerbating the burden of disease. In contrast, synergisms between species result in their becoming epidemiologically slaved within hosts, presenting a novel opportunity for controlling drug resistant parasites by targeting co-circulating species. Conclusions Understanding the effects on control of multi-parasite species interactions, and vice versa, is of increasing urgency in the advent of integrated mass intervention programmes.