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Chlamydia is the most common bacterial sexually transmitted infection worldwide and it is widely acknowledged that controlling the rampant community transmission of this infection requires vaccine development. In this study, for the first time, we elucidate the long-term response to male mouse chlamydial vaccination with chlamydial major outer membrane protein (MOMP) and ISCOMATRIX (IMX) both prophylactically and in a novel therapeutic setting. Vaccination significantly reduced and, in some cases, cleared chlamydial burden from the prostates, epididymides, and testes, which correlates with high IgG and IgA tires in tissues and serum. Important markers of sperm health and fertility were protected including sperm motility and proteins associated with fertility in men. Within splenocytes, expression of IFNγ, TNFα, IL17, IL13, IL10, and TGFβ were changed by both infection and vaccination within CD4 and CD8 T cells and regulatory T cells. Within the testicular tissue, phenotypic and concentration changes were observed in macrophages and T cells (resident and transitory). This revealed some pathogenic phenotypes associated with infection and critically that vaccination allows maintenance of testicular homeostasis, likely by preventing significant influx of CD4 T cells and promoting IL10 production. Finally, we demonstrated the testes contained immature (B220+) B cells and mature (CD138+) Chlamydia-specific plasma cells. Thus, through vaccination, we can maintain the healthy function of the testes, which is vital to protection of male fertility.

Infection with Chlamydia pecorum is one of the main causes of progressive decline of koala (Phascolarctos cinereus) populations in Eastern Australia. Pathological changes associated with the chlamydial infection in the genital tract of female and male koalas have been widely described with reports of acute and chronic lymphoplasmacytic inflammation and the description of the cystic dilatation of the ovarian bursa. Although these disease manifestations can result in severe chronic inflammation, structural changes and even sterility, only limited data is currently available on the organism's distribution and associated histopathological and ultrastructural changes within the upper genital tract of affected females. This study examined the pathogenesis of the most common pathological lesion associated with chlamydiosis in female koalas, the cystic dilation of the ovarian bursa starting from the evidence that Chlamydia spp. induces disruption of the intercellular junctions in the epithelium of the reproductive organs in humans. Histology, immunohistochemistry (IHC) and transmission electron microscopy (TEM) were performed to evaluate the structural features and the expression of epithelial cell and cellular junctions' markers in affected bursae from 39 Chlamydia-infected female koalas. Epithelial cells from the ovarian bursae of one affected animal examined by transmission electron microscopy showed severe widening of the intercellular space, as morphologic evidence of disrupted permeability of the epithelial barrier. The epithelial cell-cell junctions markers E-cadherin, β-catenin and ZO-1 expressions were significantly reduced in samples from cystic bursae when compared to normal tissue samples (P 0.0001). On the other end, a significantly higher expression of the proliferation marker Ki67 was observed in cystic bursae compared to control samples (P 0.0001). As these proteins are required to maintain epithelial functional integrity and cell-cell adhesive interactions, their loss may permanently impair and affect female koala fertility and suggest the molecular basis of the pathogenesis of the cystic accumulation of bursal fluid within this tissue.

Transmission of Chlamydia pecorum infection has generally been assumed to be via the urogenital route and in an attempt to confirm this we investigated an in vitro method of Chlamydia infection using naturally infected koala semen to inoculate a cell line and attempt to estimate C. pecorum infectious load. A total of 57% of 122 koala semen samples had low C. pecorum copy number or no burden, while 18% of semen samples contained >10 000 inclusion-forming units/mL, as determined by quantitative polymerase chain reaction. In vitro inoculation of a McCoy cell line resulted in successful infection from 4% of semen samples where C. pecorum burden was >105 inclusion-forming units/mL. Our preliminary study suggests that transmission of C. pecorum infectious dose may be restricted to peak bacterial shedding in semen associated with recent infection. Here, we report venereal transmission of C. pecorum in koala semen is possible; however, we speculate that antimicrobial factors and innate immune function receptors associated with semen may inhibit chlamydial growth. These mechanisms have yet to be reported in marsupial semen.

HSV-2 infected more than 491 million people aged 15-49 world-wide in 2016. The morbidity associated with recurrent infections and the increased risk of HIV infection make this a major health problem. To date there is no effective vaccine. Because HSV-2 ascends to the dorsal route ganglion within 12-18 hours of infection, an effective vaccine will need to elicit a strong local resident CD8+ T cell response to prevent the infection from becoming life-long.Using a mouse model we investigated the potential of oral immunization with a novel lipid adjuvant (LiporaleTM) followed by local vaginal application of an inflammatory agents to protect against primary HSV-2 infections.Oral vaccination of mice with live-attenuated HSV-2 in LiporaleActivation of mucosal immunity by oral immunization, combined with induction of transient local genital inflammation can recruit long-lived tissue resident CD8

Although it is well established that chlamydial disease renders female koalas infertile, there has been limited research on its effects on male koala fertility, specifically sperm quality. This study determined whether chlamydial infection adversely affects semen quality of naturally infected koalas and spermatozoa recovered from Chlamydia negative koalas co-incubated in vitro with C. pecorum elementary bodies (EBs). Semen from 102 south-east Queensland sexually mature wild koalas exhibiting varying degrees of chlamydiosis and clinical signs of disease were assessed for semen quality and compared to 11 clinically healthy, Chlamydia-free captive male koalas. For in vitro studies, semen samples were collected from 6 Chlamydia-free captive koalas, and co-incubated over 24 h with high and low concentrations of C. pecorum EBs and sperm quality assessed. Wild koalas displaying severe signs of clinical disease with C. pecorum present in the semen had significantly greater sperm DNA damage (P = 0.0267). The total % of morphologically abnormal spermatozoa was highest in wild koalas that had severe signs of clinical disease but whose semen was negative for C. pecorum (P = 0.0328). This apparent contradiction is possibly associated with wild males having resolved the infection but still possessing underlining reproductive pathology. A higher incidence of loose head spermatozoa occurred in semen of wild koalas not infected with C. pecorum compared to those that were C. pecorum infected (P = 0.026). In vitro incubation of semen with C. pecorum significantly decreased sperm motility and viability over 24 h.

With approximately 131 million new genital tract infections occurring each year, Chlamydia is the most common sexually transmitted bacterial pathogen worldwide. Male and female infections occur at similar rates and both cause serious pathological sequelae. Despite this, the impact of chlamydial infection on male fertility has long been debated, and the effects of paternal chlamydial infection on offspring development are unknown. Using a male mouse chronic infection model, we show that chlamydial infection persists in the testes, adversely affecting the testicular environment. Infection increased leukocyte infiltration, disrupted the blood:testis barrier and reduced spermiogenic cell numbers and seminiferous tubule volume. Sperm from infected mice had decreased motility, increased abnormal morphology, decreased zona-binding capacity, and increased DNA damage. Serum anti-sperm antibodies were also increased. When both acutely and chronically infected male mice were bred with healthy female mice, 16.7% of pups displayed developmental abnormalities. Female offspring of chronically infected sires had smaller reproductive tracts than offspring of noninfected sires. The male pups of infected sires displayed delayed testicular development, with abnormalities in sperm vitality, motility, and sperm-oocyte binding evident at sexual maturity. These data suggest that chronic testicular Chlamydia infection can contribute to male infertility, which may have an intergenerational impact on sperm quality., Testicular C. muridarum infection results in tissue damage and poor sperm quality and gives rise to offspring with serious abnormalities and developmental delays.

Most current animal vaccine regimes involve a primary vaccination followed sometime later by a booster vaccination. This presents challenges when vaccinating difficult to access animals such as livestock. Mustering livestock to deliver a vaccine boost is costly and stressful for animals. Thus, we have produced a platform system that can be administered at the same time as the priming immunisation and delivers payload after an appropriate delay time to boost the immune response, without need for further handling of animals. A 30 × 2 mm osmotically triggered polymer implant device with burst-release characteristics delivered the booster dose of a tetanus vaccine. Blood samples were collected from an experimental group that received the priming vaccine and implant on day 0 and control group that received the initial vaccine (tetanus toxoid) and then a bolus dose 28 days later via subcutaneous injection. The two groups showed identical weight gain curves. T cell proliferation following in vitro stimulation with antigen was identical between the two groups at all time points. However, serum IgG antibody responses to the tetanus toxoid antigen were significantly higher in the control group at weeks 8 and 12. The implant capsules stayed at the site of implantation and at week 12 there was evidence of tissue integration. No local reactions at the implant site were observed, other than mild thickening of the skin in half of the experimental group animals and no other adverse health events were recorded in either group.

There is growing evidence that Chlamydia pecorum infection of the male koala reproductive tract causes inflammation and pathology of the urogenital tract. Previous studies have revealed that male koalas exhibiting severe clinical signs of urogenital chlamydial disease had an increased incidence of sperm DNA fragmentation and abnormal sperm morphology, suggestive of chronic exposure to C. pecorum infection and/or inflammation in the testis and epididymis, with residual pathology and lesions disrupting spermatogenesis and maturation of spermatozoa. This study specifically aimed to determine whether pathology associated with chlamydial infection in different regions of the male koala reproductive tract had an adverse effect on classical seminal parameters, sperm DNA quality and endocrine function (testosterone secretion) of naturally infected males. Semen from 58 sexually mature male koalas deemed not suitable for rehabilitation or treatment was assessed, in addition to undertaking a GnRH challenge to determine the androgenic capacity of the testis. Following euthanasia, tissue samples from testes, epididymis and prostate were evaluated for histopathology and real time polymerase chain reaction (qPCR). A significant difference in sperm concentration was observed between males with unilateral and bilateral testicular atrophy and C. pecorum infection (P = 0.011); and between males with unilateral atrophy and C. pecorum infection in one testis and bilateral normal testes with no C. pecorum infection (P = 0.008). No significant association was found for any other semen parameters when categorised by histopathology and C. pecorum tissue presence within the testes, epididymis and prostate. Plasma testosterone concentrations did not significantly differ between testicular histopathology diagnosis and/or C. pecorum infection status. This study suggests Chlamydia infection and inflammation may not be the predominant reason of disruption to spermatogenesis in the wild koala but rather testicular degeneration and atrophy, irrespective of Chlamydia infection, appears to be the primary reason of decreased sperm concentration.

Purpose This study investigated the effect of 5 days of heat acclimation training on neuromuscular function, intestinal damage, and 20 km cycling (20TT) performance in the heat. Methods Eight recreationally trained males completed two 5-day training blocks (cycling 60 min day−1 at 50% peak power output) in a counter-balanced, cross-over design, with a 20TT completed before and after each block. Training was conducted in hot (HA: 34.9 ± 0.7 °C, 53 ± 4% relative humidity) or temperate (CON: 22.2 ± 2.6 °C, 65 ± 8% relative humidity) environment. All 20TTs were completed in the heat (35.1 ± 0.5 °C, 51 ± 4% relative humidity). Neuromuscular assessment of knee extensors (5 × 5 s maximum voluntary contraction; MVC) was completed before and after each 20TT and on the first and last days of each training block. Results MVC torque was statistically higher after 5 days of HA training compared to CON (mean difference = 14 N m [95% confidence interval; 6, 23]; p d = 0.77). However, 20TT performance after 5 days of HA training was not statistically different to CON, with a between-conditions mean difference in the completion time of 68 s [95% confidence interval; − 9, 145] (p = 0.076; d = 0.35). Conclusion Short-term heat acclimation training may increase knee extensor strength without changes in central fatigue or intestinal damage. Nevertheless, it is insufficient to improve 20 km self-paced cycling performance in the heat compared to workload-matched training in a temperate environment. These data suggest that recreationally trained athletes gain no worthwhile performance advantage from short-term heat-training before competing in the heat.

STUDY QUESTIONCan Chlamydia be found in the testes of infertile men?SUMMARY ANSWERChlamydia can be found in 16.7% of fresh testicular biopsies and 45.3% of fixed testicular biopsies taken from a selection of infertile men.WHAT IS KNOWN ALREADYMale chlamydial infection has been understudied despite male and female infections occurring at similar rates. This is particularly true of asymptomatic infections, which occur in 50% of cases. Chlamydial infection has also been associated with increased sperm DNA damage and reduced male fertility.STUDY DESIGN, SIZE, DURATIONWe collected diagnostic (fixed, n = 100) and therapeutic (fresh, n = 18) human testicular biopsies during sperm recovery procedures from moderately to severely infertile men in a cross-sectional approach to sampling.PARTICIPANTS/MATERIALS, SETTING, METHODSThe diagnostic and therapeutic biopsies were tested for Chlamydia-specific DNA and protein, using real-time PCR and immunohistochemical approaches, respectively. Serum samples matched to the fresh biopsies were also assayed for the presence of Chlamydia-specific antibodies using immunoblotting techniques.MAIN RESULTS AND THE ROLE OF CHANCEChlamydial major outer membrane protein was detected in fixed biopsies at a rate of 45.3%. This was confirmed by detection of chlamydial DNA and TC0500 protein (replication marker). C. trachomatis DNA was detected in fresh biopsies at a rate of 16.7%, and the sera from each of these three positive patients contained C. trachomatis-specific antibodies. Overall, C. trachomatis-specific antibodies were detected in 72.2% of the serum samples from the patients providing fresh biopsies, although none of the patients were symptomatic nor had they reported a previous sexually transmitted infection diagnosis including Chlamydia.LIMITATIONS, REASONS FOR CAUTIONNo reproductively healthy male testicular biopsies were tested for the presence of Chlamydia DNA or proteins or Chlamydia-specific antibodies due to the unavailability of these samples.WIDER IMPLICATIONS FOR THE FINDINGSApplication of Chlamydia-specific PCR and immunohistochemistry in this human male infertility context of testicular biopsies reveals evidence of a high prevalence of previously unrecognised infection, which may potentially have a pathogenic role in spermatogenic failure.STUDY FUNDING/COMPETING INTEREST(S)Funding for this project was provided by the Australian NHMRC under project grant number APP1062198. We also acknowledge assistance from the Monash IVF Group and Queensland Fertility Group in the collection of fresh biopsies, and the Monash Health and co-author McLachlan (declared equity interest) in retrieval and sectioning of fixed biopsies. E.M. declares an equity interest in the study due to financing of fixed biopsy sectioning. All other authors declare no conflicts of interest.TRIAL REGISTRATION NUMBERN/A

Introduction : The premise of this study was to investigate the effect of acute glutamine supplementation on 20 km time trial cycling performance in the heat, neuromuscular function, inflammation and endotoxemia. Methods : Twelve cyclists completed two, 20-km time trials (20TT) in 35 °C (50% relative humidity). Participants ingested either glutamine (GLUT; 0.9 g∙kg-1 fat-free mass) or a placebo (CON) 60 min before each 20TT. Physiological and perceptual measures were recorded during each 20TT, and neuromuscular function assessed pre- and post-exercise. Venous blood was analysed for endotoxins, markers of gut damage (inflammatory fatty acid binding protein; I-FABP) and inflammatory cytokines (interleukin-6, IL-6; tumour necrosis factor-alpha, TNF-α). Data were analysed using linear mixed models in a Bayesian framework. Results : 20TT in the heat increased I-FABP and elevated inflammatory cytokines (IL-6 and TNF-α) compared to pre-exercise values but did not result in endotoxemia. Completion time was not statistically different between conditions (mean difference [95% credible interval] = 11 s [-23, 44]). Relative to CON, GLUT did not alter any physiological or perceptual measures during the 20TT. Conclusion : Glutamine supplementation does not improve 20TT performance in the heat or preserve neuromuscular function when compared to a placebo. These findings suggest that glutamine is not an ergogenic aid or prophylactic intervention for heat-induced gut damage during short-duration self-paced exercise in hot environments.

Chlamydia infection remains the leading sexually-transmitted bacterial infection worldwide, causing damaging sequelae such as tubal scarring, infertility and ectopic pregnancy. As infection is often asymptomatic, prevention via vaccination is the optimal strategy for disease control. Vaccination strategies aimed at preventing bacterial infection have shown some promise, although these strategies often fail to prevent damaging inflammatory pathology when Chlamydia is encountered. Using a murine model of Chlamydia muridarum genital infection, we employed two established independent models to compare immune responses underpinning pathologic development of genital Chlamydia infection. Model one uses antibiotic treatment during infection, with only early treatment preventing pathology. Model two uses a plasmid-cured variant strain of C. muridarum that does not cause pathologic outcomes like the plasmid-containing wild-type counterpart. Using these infection models, contrasted by the development of pathology, we identified an unexpected role for macrophages. We observed that mice showing signs of pathology had greater numbers of activated macrophages present in the oviducts. This may have been due to early differences in macrophage activation and proinflammatory signaling leading to persistent or enhanced infection. These results provide valuable insight into the cellular mechanisms driving pathology in Chlamydia infection and contribute to the design and development of more effective vaccine strategies for protection against the deleterious sequelae of Chlamydia infection of the female reproductive tract.

Background Mucosal-associated invariant T (MAIT) cells represent a specialized lymphocyte population associated with chronic inflammatory disorders. Little is known, however, about MAIT cells in diseases of the kidney, including CKD. Methods To evaluate MAIT cells in human native kidneys with tubulointerstitial fibrosis, the hallmark of CKD, we used multicolor flow cytometry to identify, enumerate, and phenotype such cells from human kidney tissue biopsy samples, and immunofluorescence microscopy to localize these cells. We cocultured MAIT cells and human primary proximal tubular epithelial cells (PTECs) under hypoxic (1% oxygen) conditions to enable examination of mechanistic tubulointerstitial interactions. Results We identified MAIT cells (CD3+ TCR Va7.2+ CD161hi) in healthy and diseased kidney tissues, detecting expression of tissue-resident markers (CD103/CD69) on MAIT cells in both states. Tissue samples from kidneys with tubulointerstitial fibrosis had significantly elevated numbers of MAIT cells compared with either nonfibrotic samples from diseased kidneys or tissue samples from healthy kidneys. Furthermore, CD69 expression levels, also an established marker of lymphocyte activation, were significantly increased on MAIT cells from fibrotic tissue samples. Immunofluorescent analyses of fibrotic kidney tissue identified MAIT cells accumulating adjacent to PTECs. Notably, MAIT cells activated in the presence of human PTECs under hypoxic conditions (modeling the fibrotic microenvironment) displayed significantly upregulated expression of CD69 and cytotoxic molecules perforin and granzyme B; we also observed a corresponding significant increase in PTEC necrosis in these cocultures. Conclusions Our findings indicate that human tissue-resident MAIT cells in the kidney may contribute to the fibrotic process of CKD via complex interactions with PTECs.

Olfactory ensheathing cells (OECs) are often described as being present in both the peripheral and the central nervous systems (PNS and CNS). Furthermore, the olfactory nervous system glia limitans (the glial layer defining the PNS-CNS border) is considered unique as it consists of intermingling OECs and astrocytes. In contrast, the glia limitans of the rest of the nervous system consists solely of astrocytes which create a distinct barrier to Schwann cells (peripheral glia). The ability of OECs to interact with astrocytes is one reason why OECs are believed to be superior to Schwann cells for transplantation therapies to treat CNS injuries. We have used transgenic reporter mice in which glial cells express DsRed fluorescent protein to study the cellular constituents of the glia limitans. We found that the glia limitans layer of the olfactory nervous system is morphologically similar to elsewhere in the nervous system, with a similar low degree of intermingling between peripheral glia and astrocytes. We found that the astrocytic layer of the olfactory bulb is a distinct barrier to bacterial infection, suggesting that this layer constitutes the PNS-CNS immunological barrier. We also found that OECs interact with astrocytes in a similar fashion as Schwann cells in vitro. When cultured in three dimensions, however, there were subtle differences between OECs and Schwann cells in their interactions with astrocytes. We therefore suggest that glial fibrillary acidic protein-reactive astrocyte layer of the olfactory bulb constitutes the glia limitans of the olfactory nervous system and that OECs are primarily "PNS glia."

Chlamydia is the most commonly reported sexually transmitted bacterial infection, with 127 million notifications worldwide each year. Both males and females are susceptible to the pathological impacts on fertility that Chlamydia infections can induce. However, male chlamydial infections, particularly within the upper reproductive tract, including the testis, are not well characterized. In this study, using mouse testicular cell lines, we examined the impact of infection on testicular cell lineage transcriptomes and potential mechanisms for this impact. The somatic cell lineages exhibited significantly fragmented genomes during infection. Likely resulting from this, each of the Leydig, Sertoli and germ cell lineages experienced extensive transcriptional dysregulation, leading to significant changes in cellular biological pathways, including interferon and germ-Sertoli cell signalling. The cell lineages, as well as isolated spermatozoa from infected mice, also contained globally hypomethylated DNA. Cumulatively, the DNA damage and epigenetic-mediated transcriptional dysregulation observed within testicular cells during chlamydial infection could result in the production of spermatozoa with abnormal epigenomes, resulting in previously observed subfertility in infected animals and congenital defects in their offspring.

ZAP-70SKG, Il17a-deficient SKG, and BALB/c female mice were infected with C muridarum or C muridarum luciferase in the genitals. C muridarum dissemination was assessed by in vivo imaging or genomic DNA amplification. Macrophages were depleted using clodronate liposomes. Anti-tumor necrosis factor (anti-TNF) and anti-interleukin-23p19 (anti-IL-23p19) were administered after infection or arthritis onset. Gene expression of Hspa5, Tgtp1, Il23a, Il17a, Il12b, and Tnf was compared in SKG mice and BALB/c mice.One week following infection with C muridarum, macrophages and neutrophils were observed to have infiltrated the uteri of mice and were also shown to have carried C muridarum DNA to the spleen. C muridarum load was higher in SKG mice than in BALB/c mice. Macrophage depletion was shown to reduce C muridarum load and prevent development of arthritis. Compared with BALB/c mice, expression of Il23a and Il17a was increased in the uterine and splenic neutrophils of SKG mice. The presence of anti-IL-23p19 during infection or Il17a deficiency suppressed arthritis. Tnf was overexpressed in the joints of SKG mice within 1 week postinfection, and persisted beyond the first week. TNF inhibition during infection or at arthritis onset suppressed the development of arthritis. Levels of endoplasmic reticulum stress were constitutively increased in the joints of SKG mice but were induced, in conjunction with immunity-related GTPase, by C muridarum infection in the uterus.C muridarum load is higher in SKG mice than in BALB/c mice. Whereas proinflammatory IL-23 produced by neutrophils contributes to the initiation of C muridarum-mediated ReA, macrophage depletion reduces C muridarum dissemination to other tissues, tissue burden, and the development of arthritis. TNF inhibition was also shown to suppress arthritis development. Our data suggest that enhanced bacterial dissemination in macrophages of SKG mice drives the TNF production needed for persistent arthritis.

Despite the global incidence of both male infertility and sexually transmitted infections rising each year, the relationship between the two is relatively unstudied. Chlamydia is the most common bacterial sexually transmitted pathogen; however, the majority of research remains focussed on women, while the role of infection and resulting immunopathology in male factor infertility is largely unknown. Chlamydia was found in testicular biopsies from asymptomatic men with idiopathic infertility, which highlights this potential role. In animal models, testicular Chlamydia, and potentially other bacterial and viral infections, cause histopathology that is likely to adversely affect spermatogenesis and fertility. This likely occurs through infiltration of inflammatory cells, functional dysregulation of immunosuppressive testicular macrophages and Sertoli cells and destruction of key testicular cell types including sperm progenitors. Here, testicular damage due to infection and/or inflammation is reviewed, as it represents a probable underestimated and unrecognized factor leading to male infertility.

Organisms belonging to the Family Chlamydiaceae are responsible for a broad range of diseases in humans, livestock, companion animals and non-domestic species. Infection of the reproductive organs can cause a range of syndromes of which sub- and infertility are the most frequently observed clinical manifestations. While the gross and histological lesions associated with the isolation of Chlamydiaceae from the non-human female reproductive tract are well documented, little attention has been given to the pathological effects of this infection in the male genital system. As such, the occurrence and importance of Chlamydia-associated disease in male non-human mammalian species is less well documented. In order to improve our understanding of the significance of chlamydiosis in domestic, laboratory and wild animals, this review provides an up-to-date summary of Chlamydia-associated male reproductive pathology, whether that infection occurs naturally or experimentally. Although most lesions in males are described as incidental and of minor significance, results of recent studies suggest that infection with Chlamydiaceae can adversely impact male fertility and/or be instrumental in disease transmission. Although in humans, bulls and mice Chlamydia infection has been associated with morphological and functional abnormalities of the spermatozoa, this review will focus on the gross and histological findings linked to the colonisation of the genital system by this pathogen. Advances in our understanding of male reproductive chlamydiosis are necessary for diagnostic and therapeutic strategies, as well as epidemiological and conservation studies.

The infectious disease melioidosis is caused by the bacterium Burkholderia pseudomallei. Melioidosis is characterised by high mortality and morbidity and can involve the central nervous system (CNS). We have previously discovered that B. pseudomallei can infect the CNS via the olfactory and trigeminal nerves in mice. We have shown that the nerve path is dependent on mouse strain, with outbred mice showing resistance to olfactory nerve infection. Damage to the nasal epithelium by environmental factors is common, and we hypothesised that injury to the olfactory epithelium may increase the vulnerability of the olfactory nerve to microbial insult. We therefore investigated this, using outbred mice that were intranasally inoculated with B. pseudomallei, with or without methimazole-induced injury to the olfactory neuroepithelium. Methimazole-mediated injury resulted in increased B. pseudomallei invasion of the olfactory epithelium, and only in pre-injured animals were bacteria found in the olfactory nerve and bulb. In vitro assays demonstrated that B. pseudomallei readily infected glial cells isolated from the olfactory and trigeminal nerves (olfactory ensheathing cells and trigeminal Schwann cells, respectively). Bacteria were degraded by some cells but persisted in other cells, which led to the formation of multinucleated giant cells (MNGCs), with olfactory ensheathing cells less likely to form MNGCs than Schwann cells. Double Cap mutant bacteria, lacking the protein BimA, did not form MNGCs. These data suggest that injuries to the olfactory epithelium expose the primary olfactory nervous system to bacterial invasion, which can then result in CNS infection with potential pathogenic consequences for the glial cells., Author summary Infections of the central nervous system (CNS), though uncommon, are associated with severe morbidity and mortality. Burkholderia pseudomallei, the causative agent of melioidosis, can infect the CNS. We have shown that B. pseudomallei can enter the CNS via peripheral nerves extending between the nasal cavity and the brain (bypassing the blood-brain/blood-cerebrospinal fluid barriers). In the current study, we show that prior injury to the olfactory epithelium can increase B. pseudomallei invasion of the olfactory nerve and bulb, highlighting a novel risk factor for CNS infections. We also demonstrate the ability of peripheral nerve glia to internalise B. pseudomallei, resulting in the formation of multinucleated giant cells (MNGCs), dependent on the bacterial protein BimA. These findings provide important new insights into the pathogenesis of B. pseudomallei.

More than 100 million new sexually transmitted infections (STIs) are acquired every day, with 357 million new infections each year caused by chlamydia, gonorrhea, syphilis, and trichomoniasis. At least 500 million people are estimated to have a genital herpes infection. Bacterial STIs are currently treatable with antibiotics, although antibiotic-resistant gonorrhea is becoming a major problem. Antivirals can be used to reduce symptoms and shedding of genital herpes but cannot cure latent infections. To date, vaccines have been developed only against human papillomavirus and hepatitis B, despite the recognition that vaccines represent the best means of halting this hidden STI epidemic. To facilitate successful reproduction, immunity in both the female and male reproductive tracts must be regulated. In females reproductive tract, immunity is regulated by sex hormones during pregnancy to allow successful implantation and growth to term of a semiallogeneic fetus. In males, sperm development begins at puberty, well after tolerance to self has developed. Therefore immune responses in the testes and epididymides must be suppressed to prevent autoimmunity against developing sperm and male infertility. These restraints on reproductive tract immune responses not only may provide opportunities for many sexually transmitted pathogens to establish chronic infections but also represent major challenges to the development of successful vaccines to target the major STIs. In this chapter, we describe how immunity in the female and male reproductive tracts is regulated to facilitate reproduction and discuss current research progress toward the development of vaccines for chlamydia and genital herpes.

Natural killer (NK) cells are a population of lymphoid cells that play a significant role in mediating innate immune responses. Studies in mice suggest a pathological role for NK cells in models of kidney disease. In this study, we characterized the NK cell subsets present in native kidneys of patients with tubulointerstitial fibrosis, the pathological hallmark of chronic kidney disease. Significantly higher numbers of total NK cells (CD3 - CD56 + ) were detected in renal biopsies with tubulointerstitial fibrosis compared with diseased biopsies without fibrosis and healthy kidney tissue using multi-color flow cytometry. At a subset level, both the CD56 dim NK cell subset and particularly the CD56 bright NK cell subset were elevated in fibrotic kidney tissue. However, only CD56 bright NK cells significantly correlated with the loss of kidney function. Expression of the tissue-retention and -activation molecule CD69 on CD56 bright NK cells was significantly increased in fibrotic biopsy specimens compared with non-fibrotic kidney tissue, indicative of a pathogenic phenotype. Further flow cytometric phenotyping revealed selective co-expression of activating receptor CD335 (NKp46) and differentiation marker CD117 (c-kit) on CD56 bright NK cells. Multi-color immunofluorescent staining of fibrotic kidney tissue localized the accumulation of NK cells within the tubulointerstitium, with CD56 bright NK cells (NKp46 + CD117 + ) identified as the source of pro-inflammatory cytokine interferon-γ within the NK cell compartment. Thus, activated interferon-γ–producing CD56 bright NK cells are positioned to play a key role in the fibrotic process and progression to chronic kidney disease.

Group B Streptococcus (GBS) is a common colonizer of the female genital tract at the time of pregnancy, and has been associated with severe neonatal infections. Despite trials for GBS vaccines already being underway, the factors influencing vaginal GBS colonization and clearance are currently poorly understood. Within this study, we investigated the host immune responses to GBS infections in mice that affect GBS vaginal colonization and clearance. Cervicovaginal swabs were used to measure vaginal GBS persistence, and vaginal cytokine responses were measured using the Bio-Plex® system. Lymphocytes isolated from spleens were stimulated with UV-killed GBS to examine systemic cellular responses. Additional in vitro cellular experiments using human vaginal epithelial cells were also performed, examining the effect pregnancy level hormones had on GBS adhesion, invasion and cytokine responses. We observed significant differences in the ability of GBS serotype V infections to persist, compared to GBS serotype Ia vaginal infections. Vaginal cytokine response examination identified temporal changes in cytokine production (IL10, IFNγ, IL6, IL1β, TNFα) in relation to GBS serotype and clearance or colonization. Lymphocyte proliferation assays also revealed robust cellular immune responses to GBS vaginal infections irrespective of clearance or colonization. In vitro human cellular analyses also identified that vaginal epithelial cell line cytokine production was suppressed in the presence of hormones despite no alteration in adhesion/invasion. Here, we establish previously unknown, serotype specific, temporal immune responses which may be associated with vaginal GBS colonization or clearance in the female genital tract.

Infectious disease, predominately chlamydiosis, contributes significantly to the decline in health of wild koala (Phascolarctos cinereus) populations in some regions of Australia. In this study, we describe the development and evaluation of a simple, sensitive, and specific loop‐mediated isothermal amplification (LAMP) assay for the detection of Chlamydia pecorum in koalas as a point‐of‐care diagnostic tool that can be used in any wildlife hospital and in the field on specialized instrumentation. A set of primers targeting a 188‐bp region of the C. pecorum genome was designed. 100% specificity of the LAMP assay was revealed by demonstrating no cross‐reactivity with 33 nontarget pathogens, and complete correlation with qPCR results for 43 clinical swabs collected opportunistically from wildlife hospitals. In sensitivity evaluations, the technique successfully detected serial dilutions of extracted C. pecorum DNA with a detection limit of 44 IFU/ml., In this study, we describe the development and evaluation of a Chlamydia pecorum‐specific loop‐mediated isothermal amplification (LAMP) assay on the Genie ® III as a rapid point‐of care diagnostic test that can be easily incorporated into wildlife hospitals or koala field surveys. Furthermore, we present a simplistic one‐step lysis method for template preparation using koala conjunctival, urogenital, and rectal swabs. We have demonstrated the C. pecorum LAMP assay is highly sensitive and specific compared with real‐time PCR, with a level of detection of 44 C. pecorum IFU/ml.

Chlamydia spp. infections cause immunopathology of the male and female urogenital tracts and incidence continues to rise across the globe. Animal models offer the opportunity to study the host: pathogen relationship, with rodent models being an attractive first step in studying immune interactions, genetic knockout, as well as bacterial inhibitor and vaccine trials. Here we describe the methodology to infect both male and female rodents at various mucosal sites, with a particular focus on the reproductive tracts.

Non-resolved persistent macrophage-mediated synovial inflammation is considered as one of the main drivers of both the establishment and progression of obesity-associated osteoarthritis (OA). Herein, we used clodronate-loaded liposomes (CL) to locally deplete macrophages in the synovial joints to examine the role of macrophages in the progression of obesity-induced OA. Furthermore, resolvin D1 (RvD1), a unique family of pro-resolving lipid mediator derived from the omega-3 polyunsaturated fatty acid, have shown marked potency in changing the pro-inflammatory behaviour of the macrophages. We sought to determine whether RvD1 administration ameliorates obesity-induced OA by resolving macrophage-mediated synovitis. Therapeutic properties of RvD1 and macrophage depletion (CL) were tested for its ability to slow post-traumatic OA (PTOA) in obese mice models. PTOA was induced in C57Bl/6 mice fed with high-fat diet (HFD) by surgically destabilising the meniscus. Firstly, CL treatment showed beneficial effects in reducing synovitis and cartilage destruction in obese mice with PTOA. In vitro treatment with RvD1 decreased the levels of pro-inflammatory markers in CD14+ human macrophages. Furthermore, intra-articular treatment with RvD1 diminishes the progression of OA in the knee joint from mice as follows: (a) decreases macrophages infiltration in synovium, (b) reduces the number of pro-inflammatory macrophages in synovium and (c) improves the severity of synovitis and cartilage degradation. Thus, our results provide new evidence for the potential targeting of macrophages in the treatment of obesity-induced OA.

Chlamydia spp. infections cause immunopathology of the male and female urogenital tracts and incidence continues to rise across the globe. Animal models offer the opportunity to study the host: pathogen relationship, with rodent models being an attractive first step in studying immune interactions, genetic knockout, as well as bacterial inhibitor and vaccine trials. Here we describe the methodology to infect both male and female rodents at various mucosal sites, with a particular focus on the reproductive tracts.

The koala (Phascolarctos cinereus) is an arboreal herbivorous marsupial that is an Australian icon. Koalas in many parts of Australia are under multiple threats including habitat destruction, dog attacks, vehicular accidents, and infectious diseases such as Chlamydia spp. and the koala retrovirus (KoRV), which may contribute to the incidence of lymphoma and leukaemia in this species. Due to a lack of koala-specific immune reagents and assays there is currently no way to adequately analyse the immune response in healthy, diseased or vaccinated animals. This paper reports the production and characterisation of the first anti-koala CD4 monoclonal antibody (mAb). The koala CD4 gene was identified and used to develop recombinant proteins for mAb production. Fluorochrome-conjugated anti-CD4 mAb was used to measure the levels of CD4(+) lymphocytes collected from koala spleens (41.1%, range 20-45.1%) lymph nodes (36.3%, range 19-55.9%) and peripheral blood (23.8%, range 17.3-35%) by flow cytometry. Biotin-conjugated anti-CD4 mAb was used for western blot to determine an approximate size of 52 kDa for the koala CD4 molecule and used in immunohistochemistry to identify CD4(+) cells in the paracortical region and germinal centres of spleen and lymph nodes. Using the anti-CD4 mab we showed that CD4 cells from vaccinated, but not control, koalas proliferated following in vitro stimulation with UV-inactivated Chlamydia pecorum and recombinant chlamydial antigens. Since CD4(+) T cells have been shown to play a pivotal role in clearing chlamydial infection in both human and mouse infections, using this novel antibody will help determine the role CD4(+) T cells play in protection against chlamydial infection in koalas and also enhance our knowledge of how KoRV affects the koala immune system.

Sexually transmitted Chlamydia trachomatis causes infertility, and because almost 90% of infections are asymptomatic, a vaccine is required for its eradication. Mathematical modeling studies have indicated that a vaccine eliciting partial protection (non-sterilizing) may prevent Chlamydia infection transmission, if administered to both sexes before an infection. However, reducing chlamydial inoculum transmitted by males and increasing infection resistance in females through vaccination to elicit sterilizing immunity has yet to be investigated experimentally. Here we show that a partially protective vaccine (chlamydial major outer membrane protein (MOMP) and ISCOMATRIX (IMX) provided sterilizing immunity against sexual transmission between immunized mice. Immunizing male or female mice before an infection reduced chlamydial burden and disease development, but did not prevent infection. However, infection and inflammatory disease responsible for infertility were absent in 100% of immunized female mice challenged intravaginally with ejaculate collected from infected immunized males. In contrast to the sterilizing immunity generated following recovery from a previous chlamydial infection, protective immunity conferred by MOMP/IMX occurred independent of resident memory T cells. Our results demonstrate that vaccination of males or females can further protect the opposing sex, whereas vaccination of both sexes can synergize to elicit sterilizing immunity against Chlamydia sexual transmission.

The development of a vaccine is a key strategy to combat the widespread and debilitating effects of chlamydial infection in koalas. One such vaccine in development uses recombinant chlamydial major outer membrane protein (rMOMP) as an antigen and has shown promising results in several koala trials. Previous chlamydial vaccine studies, primarily in the mouse model, suggest that both cell-mediated and antibody responses will be required for adequate protection. Recently, the important protective role of antibodies has been highlighted. In our current study, we conducted a detailed analysis of the antibody-mediated immune response in koalas that are either (a) naturally-infected, and/or (b) had received an rMOMP vaccine. Firstly, we observed that naturally-infected koalas had very low levels of Chlamydia pecorum-specific neutralising antibodies. A strong correlation between low IgG total titers/neutralising antibody levels, and higher C. pecorum infection load was also observed in these naturally-infected animals. In vaccinated koalas, we showed that the vaccine was able to boost the humoral immune response by inducing strong levels of C. pecorum-specific neutralising antibodies. A detailed characterisation of the MOMP epitope response was also performed in naturally-infected and vaccinated koalas using a PepScan epitope approach. This analysis identified unique sets of MOMP epitope antibodies between naturally-infected non-protected and diseased koalas, versus vaccinated koalas, with the latter group of animals producing a unique set of specific epitope-directed antibodies that we demonstrated were responsible for the in vitro neutralisation activity. Together, these results show the importance of antibodies in chlamydial infection and immunity following vaccination in the koala.

Declining population sizes of koalas (Phascolarctos cinereus) in SE Queensland (QLD), Australia can partially be attributed to chlamydiosis, with the majority of epidemiological studies focusing on the prevalence of infection and associated pathology in female koalas, with lesser attention given to males. We aimed to explore the epidemiology of Chlamydia pecorum infection in the male urogenital tract from wild (hospitalized and free-ranging) koalas in SE QLD. Although 67% of male koalas were infected with C. pecorum in their urogenital tract and 55% were shedding the organism in their semen, only a third of the males sampled presented with overt signs of urogenital disease. Infection with C. pecorum was lower in populations from rural locations, compared with periurban locations, with a corresponding low association between urogenital infection and clinical disease. The presence of C. pecorum in penile urethral swabs was a good predictor of the presence of C. pecorum in semen, with a significant correlation (P=0.006) in 58% of males. In contrast, the C. pecorum load in penile urethral swabs was not a good predictor of the C. pecorum load in semen, with no significant correlation. In addition, 57% of male koalas had large numbers of bacterial copy numbers in the penile urethra (upper quartile) and 40% shedding into semen with no overt signs of disease. Investigation of the association of C. pecorum infection, body condition score, and age revealed that the highest incidence of urogenital infection occurred in males with the lowest body score (1 out of 10). Furthermore, 63% of sexually mature male koalas (>2 yr old) had urethral infections and 50% had C. pecorum in their semen. Our study suggested that the role of chlamydia in male koala infertility has been previously underestimated.

Chlamydiosis is the most documented and serious disease of koalas, characterized by ocular, urinary, and reproductive lesions. Since little attention has been paid to the pathological effects of this infection in the male reproductive system, we aimed to determine the incidence and severity of reproductive pathology associated with chlamydial infection in male koalas submitted to koala hospitals in southeast Queensland. The entire reproductive tract from 62 sexually mature male koalas not suitable for rehabilitation was evaluated and 677 tissue samples were collected for histology, immunohistochemistry (IHC), and real-time polymerase chain reaction (qPCR). Lymphoplasmacytic inflammation was observed in 178 of 677 (26.3%) tissue samples from the upper and lower reproductive tract, mainly in the prostatic, penile, and membranous urethra. IHC was positive for the chlamydial antigen in 19 of 451 normal samples (4.2%) and 46 of 178 samples with inflammation (25.8%), located within the cytoplasm of epithelial cells of the epididymis, vas deferens, prostate, bulbourethral glands, and the prostatic membranous and penile urethra. Chlamydia pecorum was detected via qPCR in 319 of 451 normal samples (70.7%) and 159 of 178 samples with inflammation (89.3%), with the highest incidence in the penile urethra, prostate, membranous urethra, and bulbourethral glands. This study suggests that Chlamydia infection in the male reproductive tract is more widespread than originally thought. Furthermore, the male reproductive tract might be a reservoir for persistent chlamydial infections in koalas, with important implications for prophylactic strategies and epidemiology.

Comprehensively explores sexually transmitted diseases, from epidemiology, causative pathogens, clinical impact, and immunology, to management strategies utilizing new strategies of genomics and next-generation diagnostic tools Sexually transmitted infections (STI) are very common worldwide. More than 20 different STIs have been identified, and about 19 million men and women are infected each year in the United States alone. This book looks at the complete picture of common STIs— how they form, evolve, and transmit, as well as how they can be treated and managed with modern techniques, medicines, and tools. Diagnostics to Pathogenomics of Sexually Transmitted Infections runs the spectrum of discussion ranging from introduction of causative pathogen, their pathogenesis to epidemiology, immunology, to anatomy and physiology of human genitalia and management strategies. The book offers in-depth chapter coverage on effect of probiotics on reproductive health; mucosal immunity in sexually transmitted infections; the role of circumcision in preventing STIs; Human Immunodeficiency Virus (HIV); genital herpes; molluscum contagiosum; genital warts; chlaymydia trachomatis; donovanosis; gonorrhoea; treponematoses; genital mycoplasms; bacterial vaginosis; vulvovaginal candidiasis; chlaymydia; scabies; chancroid, yeast infections; and more. - Comprehensively compiles most of the major sexually transmitted infections - Presents updated information on clinical aspects of sexually transmitted infections - Examines the priorities in pathogenesis of human sexually transmitted infections and discusses new strategies of genomics and next-generation diagnostic tools used for detection of such pathogens - Explores the future of rapid molecular diagnostic techniques and the challenges posed in the diagnosis of human STIs - Includes bench to bedside content that will appeal to both basic and clinical researchers By offering the latest knowledge about recent advances in sexually transmitted infections in an interdisciplinary fashion, Diagnostics to Pathogenomics of Sexually Transmitted Infections is the perfect book for virologists, microbiologists, infectious disease experts, vaccinologists, biomedical researchers, clinicians, pharmacologists, and public health specialists.

Infectious diseases have contributed to the decline in the health of koala ( Phascolarctos cinereus) populations in the wild in some regions of Australia. Herein we report the development and validation of 2 multiplex real-time PCR (rtPCR) panels for the simultaneous detection of Mycoplasma spp., Ureaplasma spp., Bordetella bronchiseptica, and Chlamydia, including speciation and quantification of Chlamydia, in ocular, reproductive, and nasal swab samples in addition to semen and male urogenital and reproductive tissues, from koalas. Each rtPCR panel was developed for use as a single-tube reaction using pathogen-specific primers and fluorescently labeled probe sets. DNA extracted from reference strains and isolates was used for validation of sequence gene targets for the multiplex rtPCR panels. Each panel was shown to be sensitive and specific in detecting and differentiating the bacterial pathogens. The multiplex rtPCR panels were used to screen clinical samples from free-ranging and hospitalized koalas for multiple pathogens simultaneously. The multiplex rtPCR will improve turnaround time compared to individual-pathogen rtPCR methods used, to date, for confirmation of diagnosis and will provide the wildlife clinician with the ability to make treatment decisions more rapidly.

Arthropod-borne viruses (arboviruses) are resurging across the globe. Zika virus (ZIKV) has caused significant concern in recent years because it can lead to congenital malformations in babies and Guillain-Barré syndrome in adults. Unlike other arboviruses, ZIKV can be sexually transmitted and may persist in the male reproductive tract. There is limited information regarding the impact of ZIKV on male reproductive health and fertility. Understanding the mechanisms that underlie persistent ZIKV infections in men is critical to developing effective vaccines and therapies. Mouse and macaque models have begun to unravel the pathogenesis of ZIKV infection in the male reproductive tract, with the testes and prostate gland implicated as potential reservoirs for persistent ZIKV infection. Here, we summarize current knowledge regarding the pathogenesis of ZIKV in the male reproductive tract, the development of animal models to study ZIKV infection at this site, and prospects for vaccines and therapeutics against persistent ZIKV infection.

BACKGROUND: γδ T cells are effector lymphocytes recognized as key players during chronic inflammatory processes. Mouse studies suggest a pathological role for γδ T cells in models of kidney disease. Here we evaluated γδ T cells in human native kidneys with tubulointerstitial fibrosis, the pathological hallmark of chronic kidney disease. METHODS: γδ T cells were extracted from human kidney tissue and enumerated and phenotyped by multicolour flow cytometry. Localization and cytokine production by γδ T cells was examined by immunofluorescent microscopy. RESULTS: We detected significantly elevated numbers of γδ T cells in diseased biopsies with tubulointerstitial fibrosis compared with diseased biopsies without fibrosis and healthy kidney tissue. At a subset level, only numbers of Vδ1+ γδ T cells were significantly elevated in fibrotic kidney tissue. Expression levels of cluster of differentiation 161 (CD161), a marker of human memory T cells with potential for innate-like function and interleukin (IL)-17A production, were significantly elevated on γδ T cells from fibrotic biopsies compared with nonfibrotic kidney tissue. Flow cytometric characterization of CD161+ γδ T cells in fibrotic biopsies revealed significantly elevated expression of natural killer (NK) cell-associated markers CD56, CD16 and CD336 (NKp44) compared with CD161- γδ T cells, indicative of a cytotoxic phenotype. Immunofluorescent analysis of fibrotic kidney tissue localized the accumulation of γδ T cells within the tubulointerstitium, with γδ T cells identified, for the first time, as a source of pro-inflammatory cytokine IL-17A. CONCLUSIONS: Collectively, our data suggest that human effector γδ T cells contribute to the fibrotic process and thus progression to chronic kidney disease.

The present study aimed to establish if a previously identified Chlamydia trachomatis HtrA (CtHtrA) inhibitor, JO146, is effective against currently circulating clinical isolates to validate if CtHtrA is a clinically relevant target for future therapeutic development. Inhibition of CtHtrA during the middle of the chlamydial replicative cycle until the completion of the cycle resulted in loss of infectious progeny for six unique clinical isolates representing different serovars. This supports the potential for CtHtrA to be a clinically relevant target for development of new therapeutics and suggests the importance of further investigation of JO146 as a lead compound.