In-situ fluorescence spectroscopy is a more rapid and resilient indicator of faecal contamination risk in drinking water than faecal indicator organisms

Faecal indicator organisms (FIOs) are limited in their ability to protect public health from the microbial contamination of drinking water because of their transience and time required to deliver a result. We evaluated alternative rapid, and potentially more resilient, approaches against a benchmark FIO of thermotolerant coliforms (TTCs) to characterise faecal contamination over 14 months at 40 groundwater sources in a Ugandan town. Rapid approaches included: in-situ tryptophan-like fluorescence (TLF), humic-like fluorescence (HLF), turbidity; sanitary inspections; and total bacterial cells by flow cytometry. TTCs varied widely in six sampling visits: a third of sources tested both positive and negative, 50% of sources had a range of at least 720 cfu/100 mL, and a two-day heavy rainfall event increased median TTCs five-fold. Using source medians, TLF was the best predictor in logistic regression models of TTCs ≥10 cfu/100 mL (AUC 0.88) and best correlated to TTC enumeration (ρs 0.81), with HLF performing similarly. Relationships between TLF or HLF and TTCs were stronger in the wet season than the dry season, when TLF and HLF were instead more associated with total bacterial cells. Source rank-order between sampling rounds was considerably more consistent, according to cross-correlations, using TLF or HLF (min ρs 0.81) than TTCs (min ρs 0.34). Furthermore, dry season TLF and HLF cross-correlated more strongly (ρs 0.68) than dry season TTCs (ρs 0.50) with wet season TTCs, when TTCs were elevated. In-situ TLF or HLF are more rapid and resilient indicators of faecal contamination risk than TTCs

Enhancement of outflow facility in the murine eye by targeting selected tight-junctions of Schlemm's canal endothelia

The juxtacanalicular connective tissue of the trabecular meshwork together with inner wall endothelium of Schlemm’s canal (SC) provide the bulk of resistance to aqueous outflow from the anterior chamber. Endothelial cells lining SC elaborate tight junctions (TJs), down-regulation of which may widen paracellular spaces between cells, allowing greater fluid outflow. We observed significant increase in paracellular permeability following siRNA-mediated suppression of TJ transcripts, claudin-11, zonula-occludens-1 (ZO-1) and tricellulin in human SC endothelial monolayers. In mice claudin-11 was not detected, but intracameral injection of siRNAs targeting ZO-1 and tricellulin increased outflow facility significantly. Structural qualitative and quantitative analysis of SC inner wall by transmission electron microscopy revealed significantly more open clefts between endothelial cells treated with targeting, as opposed to non-targeting siRNA. These data substantiate the concept that the continuity of SC endothelium is an important determinant of outflow resistance, and suggest that SC endothelial TJs represent a specific target for enhancement of aqueous movement through the conventional outflow system

Existing Infection Facilitates Establishment and Density of Malaria Parasites in Their Mosquito Vector

Very little is known about how vector-borne pathogens interact within their vector and how this impacts transmission. Here we show that mosquitoes can accumulate mixed strain malaria infections after feeding on multiple hosts. We found that parasites have a greater chance of establishing and reach higher densities if another strain is already present in a mosquito. Mixed infections contained more parasites but these larger populations did not have a detectable impact on vector survival. Together these results suggest that mosquitoes taking multiple infective bites may disproportionally contribute to malaria transmission. This will increase rates of mixed infections in vertebrate hosts, with implications for the evolution of parasite virulence and the spread of drug-resistant strains. Moreover, control measures that reduce parasite prevalence in vertebrate hosts will reduce the likelihood of mosquitoes taking multiple infective feeds, and thus disproportionally reduce transmission. More generally, our study shows that the types of strain interactions detected in vertebrate hosts cannot necessarily be extrapolated to vectors

An Age-Structured Extension to the Vectorial Capacity Model

Vectorial capacity and the basic reproductive number (R(0)) have been instrumental in structuring thinking about vector-borne pathogen transmission and how best to prevent the diseases they cause. One of the more important simplifying assumptions of these models is age-independent vector mortality. A growing body of evidence indicates that insect vectors exhibit age-dependent mortality, which can have strong and varied affects on pathogen transmission dynamics and strategies for disease prevention.Based on survival analysis we derived new equations for vectorial capacity and R(0) that are valid for any pattern of age-dependent (or age-independent) vector mortality and explore the behavior of the models across various mortality patterns. The framework we present (1) lays the groundwork for an extension and refinement of the vectorial capacity paradigm by introducing an age-structured extension to the model, (2) encourages further research on the actuarial dynamics of vectors in particular and the relationship of vector mortality to pathogen transmission in general, and (3) provides a detailed quantitative basis for understanding the relative impact of reductions in vector longevity compared to other vector-borne disease prevention strategies.Accounting for age-dependent vector mortality in estimates of vectorial capacity and R(0) was most important when (1) vector densities are relatively low and the pattern of mortality can determine whether pathogen transmission will persist; i.e., determines whether R(0) is above or below 1, (2) vector population growth rate is relatively low and there are complex interactions between birth and death that differ fundamentally from birth-death relationships with age-independent mortality, and (3) the vector exhibits complex patterns of age-dependent mortality and R(0) ∼ 1. A limiting factor in the construction and evaluation of new age-dependent mortality models is the paucity of data characterizing vector mortality patterns, particularly for free ranging vectors in the field

Fire Ant Decapitating Fly Cooperative Release Programs (1994–2008): Two Pseudacteon Species, P. tricuspis and P. curvatus, Rapidly Expand Across Imported Fire Ant Populations in the Southeastern United States

Natural enemies of the imported fire ants, Solenopsis invicta Buren S. richteri Forel (Hymenoptera: Formicidae), and their hybrid, include a suite of more than 20 fire ant decapitating phorid flies from South America in the genus Pseudacteon. Over the past 12 years, many researchers and associates have cooperated in introducing several species as classical or self-sustaining biological control agents in the United States. As a result, two species of flies, Pseudacteon tricuspis Borgmeier and P. curvatus Borgmeier (Diptera: Phoridae), are well established across large areas of the southeastern United States. Whereas many researchers have published local and state information about the establishment and spread of these flies, here distribution data from both published and unpublished sources has been compiled for the entire United States with the goal of presenting confirmed and probable distributions as of the fall of 2008. Documented rates of expansion were also used to predict the distribution of these flies three years later in the fall of 2011. In the fall of 2008, eleven years after the first successful release, we estimate that P. tricuspis covered about 50% of the fire ant quarantined area and that it will occur in almost 65% of the quarantine area by 2011. Complete coverage of the fire ant quarantined area will be delayed or limited by this species' slow rate of spread and frequent failure to establish in more northerly portions of the fire ant range and also, perhaps, by its preference for red imported fire ants (S. invicta). Eight years after the first successful release of P. curvatus, two biotypes of this species (one biotype occurring predominantly in the black and hybrid imported fire ants and the other occurring in red imported fire ants) covered almost 60% of the fire ant quarantined area. We estimate these two biotypes will cover almost 90% of the quarantine area by 2011 and 100% by 2012 or 2013. Strategic selection of several distributional gaps for future releases will accelerate complete coverage of quarantine areas. However, some gaps may be best used for the release of additional species of decapitating flies because establishment rates may be higher in areas without competing species

IFN-γ-Inducible Irga6 Mediates Host Resistance against Chlamydia trachomatis via Autophagy

Chlamydial infection of the host cell induces Gamma interferon (IFNγ), a central immunoprotector for humans and mice. The primary defense against Chlamydia infection in the mouse involves the IFNγ-inducible family of IRG proteins; however, the precise mechanisms mediating the pathogen's elimination are unknown. In this study, we identify Irga6 as an important resistance factor against C. trachomatis, but not C. muridarum, infection in IFNγ-stimulated mouse embryonic fibroblasts (MEFs). We show that Irga6, Irgd, Irgm2 and Irgm3 accumulate at bacterial inclusions in MEFs upon stimulation with IFNγ, whereas Irgb6 colocalized in the presence or absence of the cytokine. This accumulation triggers a rerouting of bacterial inclusions to autophagosomes that subsequently fuse to lysosomes for elimination. Autophagy-deficient Atg5−/− MEFs and lysosomal acidification impaired cells surrender to infection. Irgm2, Irgm3 and Irgd still localize to inclusions in IFNγ-induced Atg5−/− cells, but Irga6 localization is disrupted indicating its pivotal role in pathogen resistance. Irga6-deficient (Irga6−/−) MEFs, in which chlamydial growth is enhanced, do not respond to IFNγ even though Irgb6, Irgd, Irgm2 and Irgm3 still localize to inclusions. Taken together, we identify Irga6 as a necessary factor in conferring host resistance by remodelling a classically nonfusogenic intracellular pathogen to stimulate fusion with autophagosomes, thereby rerouting the intruder to the lysosomal compartment for destruction

Expansion in CD39(+) CD4(+) Immunoregulatory T Cells and Rarity of Th17 Cells in HTLV-1 Infected Patients Is Associated with Neurological Complications

HTLV-1 infection is associated with several inflammatory disorders, including the neurodegenerative condition HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It is unclear why a minority of infected subjects develops HAM/TSP. CD4(+) T cells are the main target of infection and play a pivotal role in regulating immunity to HTLV and are hypothesized to participate in the pathogenesis of HAM/TSP. the CD39 ectonucleotidase receptor is expressed on CD4(+) T cells and based on co-expression with CD25, marks T cells with distinct regulatory (CD39(+)CD25(+)) and effector (CD39(+)CD25(-)) function. Here, we investigated the expression of CD39 on CD4(+) T cells from a cohort of HAM/TSP patients, HTLV-1 asymptomatic carriers (AC), and matched uninfected controls. the frequency of CD39(+)CD4(+) T cells was increased in HTLV-1 infected patients, regardless of clinical status. More importantly, the proportion of the immunostimulatory CD39(+)CD25(-) CD4+ T-cell subset was significantly elevated in HAM/TSP patients as compared to AC and phenotypically had lower levels of the immunoinhibitory receptor, PD-1. We saw no difference in the frequency of CD39(+)CD25(+) regulatory (Treg) cells between AC and HAM/TSP patients. However, these cells transition from being anergic to displaying a polyfunctional cytokine response following HTLV-1 infection. CD39(-)CD25(+) T cell subsets predominantly secreted the inflammatory cytokine IL-17. We found that HAM/TSP patients had significantly fewer numbers of IL-17 secreting CD4(+) T cells compared to uninfected controls. Taken together, we show that the expression of CD39 is upregulated on CD4(+) T cells HAM/TSP patients. This upregulation may play a role in the development of the proinflammatory milieu through pathways both distinct and separate among the different CD39 T cell subsets. CD39 upregulation may therefore serve as a surrogate diagnostic marker of progression and could potentially be a target for interventions to reduce the development of HAM/TSP.National Institute of Allergies and Infectious DiseasesNational Institutes of HealthUniversity of CaliforniaSan Francisco-Gladstone Institute of Virology & Immunology Center for AIDS ResearchFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)John E. Fogarty International CenterNational Center for Research ResourcesNational Institute of General Medical Sciences from the National Institutes of HealthUniv Calif San Francisco, Dept Med, Div Expt Med, San Francisco, CA 94143 USAUniv Hawaii, John A Burns Sch Med, Dept Trop Med, Hawaii Ctr AIDS, Honolulu, HI 96822 USAUniv São Paulo, Sch Med, Deparment Infect Dis, São Paulo, BrazilUniv São Paulo, Sch Med, Div Clin Immunol & Allergy, São Paulo, BrazilFuncacao Prosangue, Hemoctr São Paulo, Mol Biol Lab, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Translat Med, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Translat Med, São Paulo, BrazilSan Francisco-Gladstone Institute of Virology & Immunology Center for AIDS Research: P30 AI027763FAPESP: 04/15856-9/KallasFAPESP: 2010/05845-0/KallasFAPESP: 11/12297-2/SanabaniJohn E. Fogarty International Center: D43 TW00003National Center for Research Resources: 5P20RR016467-11National Institute of General Medical Sciences from the National Institutes of Health: 8P20GM103466-11Web of Scienc

Age-structured gametocyte allocation links immunity to epidemiology in malaria parasites

Background Despite a long history of attempts to model malaria epidemiology, the over-riding conclusion is that a detailed understanding of host-parasite interactions leading to immunity is required. It is still not known what governs the duration of an infection and how within-human parasite dynamics relate to malaria epidemiology. Presentation of the hypothesis Immunity to Plasmodium falciparum develops slowly and requires repeated exposure to the parasite, which thus generates age-structure in the host-parasite interaction. An age-structured degree of immunity would present the parasite with humans of highly variable quality. Evolutionary theory suggests that natural selection will mould adaptive phenotypes that are more precise (less variant) in "high quality" habitats, where lifetime reproductive success is best. Variability in malaria parasite gametocyte density is predicted to be less variable in those age groups who best infect mosquitoes. Thus, the extent to which variation in gametocyte density is a simple parasite phenotype reflecting the complex within-host parasite dynamics is addressed. Testing the hypothesis Gametocyte densities and corresponding infectiousness to mosquitoes from published data sets and studies in both rural and urban Cameroon are analysed. The mean and variation in gametocyte density according to age group are considered and compared with transmission success (proportion of mosquitoes infected). Across a wide range of settings endemic for malaria, the age group that infected most mosquitoes had the least variation in gametocyte density, i.e. there was a significant relationship between the variance rather than the mean gametocyte density and age-specific parasite transmission success. In these settings, the acquisition of immunity over time was evident as a decrease in asexual parasite densities with age. By contrast, in an urban setting, there were no such age-structured relationships either with variation in gametocyte density or asexual parasite density. Implications of the hypothesis Gametocyte production is seemingly predicted by evolutionary theory, insofar as a reproductive phenotype (gametocyte density) is most precisely expressed (i.e. is most invariant) in the most infectious human age group. This human age group would thus be expected to be the habitat most suitable for the parasite. Comprehension of the immuno-epidemiology of malaria, a requisite for any vaccine strategies, remains poor. Immunological characterization of the human population stratified by parasite gametocyte allocation would be a step forward in identifying the salient immunological pathways of what makes a human a good habitat

IL-2 Immunotherapy to Recently HIV-1 Infected Adults Maintains the Numbers of IL-17 Expressing CD4+ T (TH17) Cells in the Periphery

Little is known about the manipulation of IL-17 producing CD4+ T cells (TH17) on a per-cell basis in humans in vivo. Previous studies on the effects of IL-2 on IL-17 secretion in non-HIV models have shown divergent results. We hypothesized that IL-2 would mediate changes in IL-17 levels among recently HIV-1-infected adults receiving anti-retroviral therapy. We measured cytokine T cell responses to CD3/CD28, HIV-1 Gag, and CMV pp65 stimulation, and changes in multiple CD4+ T cell subsets. Those who received IL-2 showed a robust expansion of naive and total CD4+ T cell counts and T-reg counts. However, after IL-2 treatment, the frequency of TH17 cells declined, while counts of TH17 cells did not change due to an expansion of the CD4+ naïve T cell population (CD27+CD45RA+). Counts of HIV-1 Gag-specific T cells declined modestly, but CMV pp65 and CD3/CD28 stimulated populations did not change. Hence, in contrast with recent studies, our results suggest IL-2 is not a potent in vivo regulator of TH17 cell populations in HIV-1 disease. However, IL-2-mediated T-reg expansions may selectively reduce responses to certain antigen-specific populations, such as HIV-1 Gag

Infection of Anopheles gambiae mosquitoes with entomopathogenic fungi: effect of host age and blood-feeding status

Physiological characteristics of insects can influence their susceptibility to fungal infection of which age and nutritional status are among the most important. An understanding of host–pathogen interaction with respect to these physiological characteristics of the host is essential if we are to develop fungal formulations capable of reducing malaria transmission under field conditions. Here, two independent bioassays were conducted to study the effect of age and blood-feeding status on fungal infection and survival of Anopheles gambiae s.s. Giles. Mosquitoes were exposed to 2 × 1010 conidia m−2 of oil-formulated Metarhizium anisopliae ICIPE-30 and of Beauveria bassiana I93-825, respectively, and their survival was monitored daily. Three age groups of mosquitoes were exposed, 2–4, 5–8, and 9–12 days since emergence. Five groups of different feeding status were exposed: non-blood-fed, 3, 12, 36, and 72 h post-blood feeding. Fungal infection reduced the survival of mosquitoes regardless of their age and blood-feeding status. Although older mosquitoes died relatively earlier than younger ones, age did not tend to affect mosquito susceptibility to fungal infection. Non-blood-fed mosquitoes were more susceptible to fungus infection compared to all categories of blood-fed mosquitoes, except for those exposed to B. bassiana 72 h post-blood feeding. In conclusion, formulations of M. anisopliae and B. bassiana can equally affect mosquitoes of different age classes, with them being relatively more susceptible to fungus infection when non-blood-fed