Implications of faecal indicator bacteria for the microbiological assessment of roof harvested rainwater quality in Southeast Queensland, Australia

The study aimed to evaluate the suitability of Escherichia coli, enterococci and C. perfringens to assess the microbiological quality of roof harvested rainwater, and to assess whether the concentrations of these faecal indicators can be used to predict the presence or absence of specific zoonotic bacterial or protozoan pathogens. From a total of 100 samples tested, respectively 58%, 83% and 46% of samples were found to be positive for E. coli, enterococci and C. perfringens spores, as determined by traditional culture based methods. Additionally, in the samples tested, 7%, 19%, 1%, 8%, 17%, and 15% were PCR positive for A. hydrophila lip, C. coli ceuE, C. jejuni mapA, L. pneumophila mip, Salmonella invA, and G. lamblia β-giardin genes. However, none of the samples was positive for E. coli O157 LPS, VT1, VT2 and C. parvum COWP genes. The presence or absence of these potential pathogens did not correlate with any of the faecal indicator bacterial concentrations as determined by a binary logistic regression model. The roof-harvested rainwater samples tested in this study appear to be of poor microbiological quality and no significant correlation was found between the concentration of faecal indicators and pathogenic microorganisms. The use of faecal indicator bacteria raises questions regarding their reliability in assessing the microbiological quality of water and particularly their poor correlation with pathogenic microorganisms. The presence of one or more zoonotic pathogens suggests that the microbiological analysis of water should be performed, and appropriate treatment measures should be undertaken especially in tanks where the water is used for drinking.

Human and bovine adenoviruses for the detection of source-specific fecal pollution in coastal waters in Australia

In this study, the host-specificity and -sensitivity of human- and bovine-specific adenoviruses (HS-AVs and BS-AVs) were evaluated by testing wastewater/fecal samples from various animal species in Southeast, Queensland, Australia. The overall specificity and sensitivity of the HS-AVs marker were 1.0 and 0.78, respectively. These figures for the BS-AVs were 1.0 and 0.73, respectively. Twenty environmental water samples were colleted during wet conditions and 20 samples were colleted during dry conditions from the Maroochy Coastal River and tested for the presence of fecal indicator bacteria (FIB), host-specific viral markers, zoonotic bacterial and protozoan pathogens using PCR/qPCR. The concentrations of FIB in water samples collected after wet conditions were generally higher compared to dry conditions. HS-AVs was detected in 20% water samples colleted during wet conditions and whereas BS-AVs was detected in both wet (i.e., 10%) and dry (i.e., 10%) conditions. Both, C. jejuni mapA and Salmonella invA genes were detected in 10% and 10% of samples, respectively collected during dry conditions. The concentrations of Salmonella invA ranged between 3.5 × 102 to 4.3 × 102 genomic copies per 500 ml of water G. lamblia β-giardin gene was detected only in one sample (5%) collected during the dry conditions. Weak or significant correlations were observed between FIB with viral markers and zoonotic pathogens. However, during dry conditions, no significant correlations were observed between FIB concentrations with viral markers and zoonotic pathogens. The prevalence of HS-AVs in samples collected from the study river suggests that the quality of water is affected by human fecal pollution and as well as bovine fecal pollution. The results suggest that HS-AVs and BS-AVs detection using PCR could be a useful tool for the identification of human sourced fecal pollution in coastal waters.

Health risk from the use of roof-harvested rainwater in Southeast Queensland, Australia, as potable or nonpotable water, determined using quantitative microbial risk assessment

A total of 214 rainwater samples from 82 tanks were collected in urban Southeast Queensland (SEQ) in Australia and analysed for the zoonotic bacterial and protozoan pathogen using real-time binary PCR and quantitative PCR (qPCR). Quantitative Microbial Risk Assessment (QMRA) analysis was used to quantify the risk of infection associated with the exposure to potential pathogens from potable and non-potable uses of roof-harvested rainwater. Of the 214 samples tested, 10.7%, 9.8%, and 5.6%, and 0.4% samples were positive for Salmonella invA, Giardia lamblia β-giardin , Legionella pneumophila mip, and Campylobacter jejuni mapA genes. Cryptosporidium parvum could not be detected. The estimated numbers of viable Salmonella spp., G. lamblia β-giradin, and L. pneumophila genes ranged from 1.6 × 101 to 9.5 × 101 cells, 1.4 × 10-1 to 9.0 × 10-1 cysts, and 1.5 × 101 to 4.3 × 101 per 1000 ml of water, respectively. Six risk scenarios were considered from exposure to Salmonella spp., G. lamblia and L. pneumophila. For Salmonella spp., and G. lamblia, these scenarios were: (1) liquid ingestion due to drinking of rainwater on a daily basis (2) accidental liquid ingestion due to garden hosing twice a week (3) aerosol ingestion due to showering on a daily basis, and (4) aerosol ingestion due to hosing twice a week. For L. pneumophila, these scenarios were: (5) aerosol inhalation due to showering on a daily basis, and (6) aerosol inhalation due to hosing twice a week. The risk of infection from Salmonella spp., G. lamblia, and L. pneumophila associated with the use of rainwater for showering and garden hosing was calculated to be well below the threshold value of one extra infection per 10,000 persons per year in urban SEQ. However, the risk of infection from ingesting Salmonella spp. and G. lamblia via drinking exceeds this threshold value, and indicates that if undisinfected rainwater were ingested by drinking, then the gastrointestinal diseases of Salmonellosis and Giardiasis is expected to range from 5.0 × 100 to 2.8 × 101 (Salmonellosis) and 1.0 × 101 to 6.4 × 101 (Giardiasis) cases per 10,000 persons per year, respectively. Since this health risk seems higher than that expected from the reported incidences of gastroenteritis, the assumptions used to estimate these infection risks are critically examined. Nonetheless, it would seem prudent to disinfect rainwater for potable use.

Identification of novel markers for uncomplicated lower genital tract infections and upper genital tract pathology due to Chlamydia trachomatis.

Background: Untreated Chlamydia trachomatis infections in women can result in disease sequelae such as salpingitis and pelvic inflammatory disease (PID), ultimately culminating in tubal occlusion and infertility. Whilst nucleic acid amplification tests can effectively diagnose uncomplicated lower genital tract (LGT) infections, they are not suitable for diagnosing upper genital tract (UGT) pathological sequelae. As a consequence, this study aimed to identify serological markers that can, with a high degree of sensitivity and specificity, discriminate between LGT infections and UGT pathology. Methods: Plasma was collected from 73 women with a history of LGT infection, UGT pathology due to C. trachomatis or no serological evidence of C. trachomatis infection. Western blotting was used to analyse antibody reactivity against extracted chlamydial proteins. Sensitivity and specificity of differential markers were also calculated. Results: Four antigens (CT157, CT423, CT727 and CT396) were identified and found to be capable of discriminating between the infection and disease sequelae state. Sensitivity and specificity calculations showed that our assay for diagnosing LGT infection had a sensitivity and specificity of 75% and 76% respectively, whilst the assay for identifying UGT pathology demonstrated 80% sensitivity and 86% specificity. Conclusions: The use of these assays could potentially facilitate earlier diagnoses in women suffering UGT pathology due to C. trachomatis.

Transcutaneous immunization with novel lipid-based adjuvants induces protection against gastric Helicobacter pylori infection

The development of vaccines to combat pathogens that infect across mucosal surfaces has been a major goal of vaccine research. Successful mucosal vaccination requires the co-administration of adjuvants that can overcome the state of immune tolerance normally associated with mucosal application of proteins. In the case of oral immunization, delivery systems are also required to protect vaccine antigens against destruction by gastric pH and digestive enzymes. Furthermore, adjuvants used for mucosal delivery must be free of neurotoxic effects like those induced by the commonly used experimental mucosal adjuvant cholera toxin. Maintenance of the "cold chain" is also essential for the effectiveness of any vaccine and adjuvants/delivery systems that enhance the stability of a vaccine would offer a significant advantage. Needle-free methods of vaccination that induce protective immunity at multiple mucosal surfaces are also desirable for rapid vaccination of large populations. In the present study we show that transcutaneous immunization (TCI) using Lipid C, a novel lipid-based matrix originally developed for oral immunization, containing soluble Helicobacter sonicate significantly reduces the gastric bacterial burden in mice following gastric challenge with live Helicobacter pylori. Protection is associated with the production of splenic gamma interferon and gastric IgA and was achieved without the co-administration of potent and potentially toxic adjuvants, although protection was further enhanced by inclusion of CpG-ODN and cholera toxin in the lipid delivery system.

Modeling prostate cancer: a perspective on transgenic mouse models

Despite considerable success in treatment of early stage localized prostate cancer (PC), acute inadequacy of late stage PC treatment and its inherent heterogeneity poses a formidable challenge. Clearly, an improved understanding of PC genesis and progression along with the development of new targeted therapies are warranted. Animal models, especially, transgenic immunocompetent mouse models, have proven to be the best ally in this respect. A series of models have been developed by modulation of expression of genes implicated in cancer-genesis and progression; mainly, modulation of expression of oncogenes, steroid hormone receptors, growth factors and their receptors, cell cycle and apoptosis regulators, and tumor suppressor genes have been used. Such models have contributed significantly to our understanding of the molecular and pathological aspects of PC initiation and progression. In particular, the transgenic mouse models based on multiple genetic alterations can more accurately address the inherent complexity of PC, not only in revealing the mechanisms of tumorigenesis and progression but also for clinically relevant evaluation of new therapies. Further, with advances in conditional knockout technologies, otherwise embryonically lethal gene changes can be incorporated leading to the development of new generation transgenics, thus adding significantly to our existing knowledge base. Different models and their relevance to PC research are discussed.

Inactivation and structural changes of E. Cloacae and B. Subtilis Endospores during IR laser water treatment

IR radiation has been studied for micro-organism inactivation of bacterial spores on metal substrates [1] and on metal and paper substrates [2]. A near-point near infrared laser water treatment apparatus for use in dental hand-pieces was also developed [3]. To date water sterilisation research using a mid-IR laser technique is very rare. According to the World Health Organisation [4], examinations for faecal indicator bacteria remain the most sensitive and specific way of assessing the hygienic quality of water. Bacteria that fall into this group are E. coli, other coliform bacteria (including E. cloacae) and to a lesser extent, faecal streptococci [5]. Protozoan cysts from organisms which cause giardiasis are the most frequently identified cause of waterborne diseases in developed countries [6,7]. The use of aerobic bacterial endospores to monitor the efficiency of various water treatments has been shown to provide a reliable and simple indicator of overall performance of water treatment[8,9].The efficacy of IR radiation for water disinfection compared to UV treatment has been further investigated in the present study. In addition FTIR spectroscopy in conjunction with Principle Component Analysis was used to characterise structural changes within the bacterial cells and endospores following IR laser treatment. Changes in carbohydrate content of E. cloacae following IR laser treatment were observed.

Vaccination of healthy and diseased koalas (Phascolarctos cinereus) with a Chlamydia pecorum multi-subunit vaccine : evaluation of immunity and pathology

Chlamydial infections represent a major threat to the long-term survival of the koala and a successful vaccine would provide a valuable management tool. Vaccination however has the potential to enhance inflammatory disease in animals exposed to a natural infection prior to vaccination, a finding in early human and primate trials of whole cell vaccines to prevent trachoma. In the present study, we vaccinated both healthy koalas as well as clinically diseased koalas with a multi-subunit vaccine consisting of Chlamydia pecorum MOMP and NrdB mixed with immune stimulating complex as adjuvant. Following vaccination, there was no increase in inflammatory pathological changes in animals previously infected with Chlamydia. Strong antibody (including neutralizing antibodies) and lymphocyte proliferation responses were recorded in all vaccinated koalas, both healthy and clinically diseased. Vaccine induced antibodies specific for both vaccine antigens were observed not only in plasma but also in ocular secretions. Our data shows that an experimental chlamydial vaccine is safe to use in previously infected koalas, in that it does not worsen infection-associated lesions. Furthermore, the prototype vaccine is effective, as demonstrated by strong levels of neutralizing antibody and lymphocyte proliferation responses in both healthy and clinically diseased koalas. Collectively, this work illustrates the feasibility of developing a safe and effective Chlamydia vaccine as a tool for management of disease in wild koalas.

Human Kallikrein 4 signal peptide induces cytotoxic T cell responses in healthy donors and prostate cancer patients.

Immunotherapy is a promising new treatment for patients with advanced prostate and ovarian cancer, but its application is limited by the lack of suitable target antigens that are recognized by CD8+ cytotoxic T lymphocytes (CTL). Human kallikrein 4 (KLK4) is a member of the kallikrein family of serine proteases that is significantly overexpressed in malignant versus healthy prostate and ovarian tissue, making it an attractive target for immunotherapy. We identified a naturally processed, HLA-A*0201-restricted peptide epitope within the signal sequence region of KLK4 that induced CTL responses in vitro in most healthy donors and prostate cancer patients tested. These CTL lysed HLA-A*0201+ KLK4 + cell lines and KLK4 mRNA-transfected monocyte-derived dendritic cells. CTL specific for the HLA-A*0201-restricted KLK4 peptide were more readily expanded to a higher frequency in vitro compared to the known HLA-A*0201-restricted epitopes from prostate cancer antigens; prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA) and prostatic acid phosphatase (PAP). These data demonstrate that KLK4 is an immunogenic molecule capable of inducing CTL responses and identify it as an attractive target for prostate and ovarian cancer immunotherapy.

Plant-produced vaccines: promise and reality

Plant-produced vaccines are a much-hyped development of the past two decades, whose time to embrace reality may have finally come. Vaccines have been developed against viral, bacterial, parasite and allergenic antigens, for humans and for animals; a wide variety of plants have been used for stable transgenic expression as well as for transient expression via Agrobacterium tumefaciens and plant viral vectors. A great many products have shown significant immunogenicity; several have shown efficacy in target animals or in animal models. The realised potential of plant-produced vaccines is discussed, together with future prospects for production and registration. © 2008 Elsevier Ltd. All rights reserved.

High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector

We constructed a novel autonomously replicating gene expression shuttle vector, with the aim of developing a system for transiently expressing proteins at levels useful for commercial production of vaccines and other proteins in plants. The vector, pRIC, is based on the mild strain of the geminivirus Bean yellow dwarf virus (BeYDV-m) and is replicationally released into plant cells from a recombinant Agrobacterium tumefaciens Ti plasmid. pRIC differs from most other geminivirus-based vectors in that the BeYDV replication-associated elements were included in cis rather than from a co-transfected plasmid, while the BeYDV capsid protein (CP) and movement protein (MP) genes were replaced by an antigen encoding transgene expression cassette derived from the non-replicating A. tumefaciens vector, pTRAc. We tested vector efficacy in Nicotiana benthamiana by comparing transient cytoplasmic expression between pRIC and pTRAc constructs encoding either enhanced green fluorescent protein (EGFP) or the subunit vaccine antigens, human papillomavirus subtype 16 (HPV-16) major CP L1 and human immunodeficiency virus subtype C p24 antigen. The pRIC constructs were amplified in planta by up to two orders of magnitude by replication, while 50% more HPV-16 L1 and three- to seven-fold more EGFP and HIV-1 p24 were expressed from pRIC than from pTRAc. Vector replication was shown to be correlated with increased protein expression. We anticipate that this new high-yielding plant expression vector will contribute towards the development of a viable plant production platform for vaccine candidates and other pharmaceuticals. © 2009 Blackwell Publishing Ltd.

Comparison of cervical and blood T-cell responses to human papillomavirus-16 in women with human papillomavirus-associated cervical intraepithelial neoplasia

Human papillomaviruses (HPVs) are obligate epithelial pathogens and typically cause localized mucosal infections. We therefore hypothesized that T-cell responses to HPV antigens would be greater at sites of pathology than in the blood. Focusing on HPV-16 because of its association with cervical cancer, the magnitude of HPV-specific T-cell responses at the cervix was compared with those in the peripheral blood by intracellular cytokine staining following direct ex vivo stimulation with both virus-like particles assembled from the major capsid protein L1, and the major HPV oncoprotein, E7. We show that both CD4 + and CD8 + T cells from the cervix responded to the HPV-16 antigens and that interferon-γ (IFN-γ) production was HPV type-specific. Comparing HPV-specific T-cell IFN-γ responses at the cervix with those in the blood, we found that while CD4 + and CD8 + T-cell responses to L1 were significantly correlated between compartments (P = 0.02 and P = 0.05, respectively), IFN-γ responses in both T-cell subsets were significantly greater in magnitude at the cervix than in peripheral blood (P = 0.02 and P = 0.003, respectively). In contrast, both CD4 + and CD8 + T-cell IFN-γ responses to E7 were of similar magnitude in both compartments and CD8 + responses were significantly correlated between these distinct immunological compartments (P = 0.04). We therefore show that inflammatory T-cell responses against L1 (but not E7) demonstrate clear compartmental bias and the magnitude of these responses do reflect local viral replication but that correlation of HPV-specific responses between compartments indicates their linkage.

Human papillomavirus vaccines in plants

Human papillomaviruses are the etiological agents of cervical cancer, one of the two most prevalent cancers in women in developing countries. Currently available prophylactic vaccines are based on the L1 major capsid protein, which forms virus-like particles when expressed in yeast and insect cell lines. Despite their recognized efficacy, there are significant shortcomings: the vaccines are expensive, include only two oncogenic virus types, are delivered via intramuscular injection and require a cold chain. Plant expression systems may provide ways of overcoming some of these problems, in particular the expense. In this article, we report recent promising advances in the production of prophylactic and therapeutic vaccines against human papillomavirus by expression of the relevant antigens in plants, and discuss future prospects for the use of such vaccines. © 2010 Expert Reviews Ltd.