Exploiting the potential of Insects for in vivo pathogenicity testing of Microbial pathogen

Conventional assays for quantifying the virulence of microbial pathogens and mutants have traditionally relied upon the use of a range of mammalian species. A number of workers have demonstrated that insects can be used for evaluating microbial pathogenicity and provide results comparable to those that can be obtained with mammals since one component of the vertebrate immune system, the innate immune response, remains similar to that found in insects. Larvae of the Greater Wax Moth Galleria mellonella have been used to evaluate the virulence of a range of bacterial and fungal pathogens and a correlation with the virulence of these microbes in mice has been established. This review highlights the similarities of the vertebrate and insect innate immune responses to infection and identifies the potential use of insects for the in vivo evaluation of the microbial pathogenicity

Exploiting the potential of Insects for in vivo pathogenicity testing of Microbial pathogen

Conventional assays for quantifying the virulence of microbial pathogens and mutants have traditionally relied upon the use of a range of mammalian species. A number of workers have demonstrated that insects can be used for evaluating microbial pathogenicity and provide results comparable to those that can be obtained with mammals since one component of the vertebrate immune system, the innate immune response, remains similar to that found in insects. Larvae of the Greater Wax Moth Galleria mellonella have been used to evaluate the virulence of a range of bacterial and fungal pathogens and a correlation with the virulence of these microbes in mice has been established. This review highlights the similarities of the vertebrate and insect innate immune responses to infection and identifies the potential use of insects for the in vivo evaluation of the microbial pathogenicity

Inhibition of neutrophil function following exposure to the Aspergillus fumigatus toxin fumagillin

The filamentous fungus Aspergillus fumigatus produces a variety of enzymes and toxins that may facilitate fungal colonization of tissue and evasion of the host immune response. One such toxin, fumagillin, was investigated for its ability to inhibit the action of neutrophils, which are a central component of the innate immune response to microbial infection. Neutrophils exposed to 2 mg fumagillin ml”1 for 25 min showed a significantly reduced ability to kill yeast cells (P,0.02), to phagocytose conidia of A. fumigatus (P,0.023) and to consume oxygen (P,0.032). The ability of neutrophils to generate superoxide is dependent upon the action of a functional NADPH oxidase complex which is composed of cytosolic (p40phox, p47phox, p67phox, Rac2) and membrane (gp91phox) proteins. Exposure of neutrophils to fumagillin inhibited the formation of the NADPH oxidase complex by blocking the translocation of p47phox from the cytosolic to the membrane fraction (P50.02). In addition to the production of superoxide, neutrophils also undergo degranulation, which leads to the release of proteolytic enzymes that contribute to the microbicidal activity of the cell. Fumagillin-treated neutrophils showed reduced degranulation as evidenced by lower myeloperoxidase activity (P,0.019). Fumagillin-treated cells demonstrated reduced levels of F-actin, thus indicating that retarding the formation of F-actin may contribute to the inhibition of the structural rearrangements required in the activated neutrophil. This work indicates that fumagillin may contribute to reducing the local immune response by altering the activity of neutrophils and thus facilitate the continued persistence and growth of A. fumigatus in the host

The Aspergillus fumigatus toxin fumagillin suppresses the immune response of Galleria mellonella larvae by inhibiting the action of haemocytes

Larvae of Galleria mellonella are widely used to evaluate microbial virulence and to assess the in vivo efficacy of antimicrobial agents. The aim of this work was to examine the ability of an Aspergillus fumigatus toxin, fumagillin, to suppress the immune response of larvae. Administration of fumagillin to larvae increased their susceptibility to subsequent infection with A. fumigatus conidia (P50.0052). It was demonstrated that a dose of 2 mg fumagillin ml”1 reduced the ability of insect immune cells (haemocytes) to kill opsonized cells of Candida albicans (P50.039) and to phagocytose A. fumigatus conidia (P50.016). Fumagillin reduced the oxygen uptake of haemocytes and decreased the translocation of a p47 protein which is homologous to p47phox, a protein essential for the formation of a functional NADPH oxidase complex required for superoxide production. In addition, toxin-treated haemocytes showed reduced levels of degranulation as measured by the release of a protein showing reactivity to an anti-myeloperoxidase antibody (P,0.049) that was subsequently identified by liquid chromatography-MS analysis as prophenoloxidase. This work demonstrates that fumagillin suppresses the immune response of G. mellonella larvae by inhibiting the action of haemocytes and thus renders the larvae susceptible to infection. During growth of the fungus in the larvae, this toxin, along with others, may facilitate growth by suppressing the cellular immune response

Inhibition of neutrophil function following exposure to the Aspergillus fumigatus toxin fumagillin

The filamentous fungus Aspergillus fumigatus produces a variety of enzymes and toxins that may facilitate fungal colonization of tissue and evasion of the host immune response. One such toxin, fumagillin, was investigated for its ability to inhibit the action of neutrophils, which are a central component of the innate immune response to microbial infection. Neutrophils exposed to 2 mg fumagillin ml”1 for 25 min showed a significantly reduced ability to kill yeast cells (P,0.02), to phagocytose conidia of A. fumigatus (P,0.023) and to consume oxygen (P,0.032). The ability of neutrophils to generate superoxide is dependent upon the action of a functional NADPH oxidase complex which is composed of cytosolic (p40phox, p47phox, p67phox, Rac2) and membrane (gp91phox) proteins. Exposure of neutrophils to fumagillin inhibited the formation of the NADPH oxidase complex by blocking the translocation of p47phox from the cytosolic to the membrane fraction (P50.02). In addition to the production of superoxide, neutrophils also undergo degranulation, which leads to the release of proteolytic enzymes that contribute to the microbicidal activity of the cell. Fumagillin-treated neutrophils showed reduced degranulation as evidenced by lower myeloperoxidase activity (P,0.019). Fumagillin-treated cells demonstrated reduced levels of F-actin, thus indicating that retarding the formation of F-actin may contribute to the inhibition of the structural rearrangements required in the activated neutrophil. This work indicates that fumagillin may contribute to reducing the local immune response by altering the activity of neutrophils and thus facilitate the continued persistence and growth of A. fumigatus in the host

Ex-post decomposition analysis of passenger car energy demand and associated CO2 emissions

This paper investigates, quantifies and ranks the factors influencing passenger cars energy demand and emissions. A vehicle stock-model approach is used for an ex-post decomposition analysis, based on administrative data, examining the impact seven underlying factors driving energy demand. The impact of methodological choice and model disaggregation are also explored. In light of the 2015 vehicle emissions scandal, the paper quantifies the difference between manufacturer-test vehicle performance and real world or â on-roadâ performance for a national stock model and determines the relative impact on passenger cars energy consumption. When examining the technical performance improvement, the choice of metric can lead to a distortion of 2.2 percentage points (14% overestimate) in the quantification of the efficiency improvement of the vehicle stock. The analysis pays particular attention to the influence of fuel or technology switching â which is often quoted as a factor influencing energy use and emissions but rarely quantified. Even when using litres per hundred-kilometre gasoline equivalent to measure the performance improvement, changes in the makeup of the stock can lead to distortion in the efficiency measure. The results of a full decomposition analysis highlight that technical performance improvements (energy efficiency improvements for the purpose of this paper), will not provide significant energy and emission savings when the impact on-road consumption is included. The paper concludes that technology switching in conjunction with policies targeting ownership and usage are the most effective measures to control passenger car energy consumption and associated CO2 emissions

Quantifying transport energy efficiency savings

The importance of quantifying energy savings and improvement in energy efficiency for each sector of the economy is now widely recognized in order to demonstrate progress towards targets and compliance with legal obligations. The focus of this paper is specifically on evaluating energy efficiency in transport using the ODEX methodology. More detailed data has recently become available on transport energy trends and the underlying factors that allow the authors improve the calculation of Ireland’s transport ODEX. Through data mining of administrative databases mileage, volume, age, engine type and size data are available at a disaggregated level for each mode of road transport. In particular this paper examines private car energy efficiency, quantifying the change arising from improved data. There was an overall slight improvement (0.71 percentage points) in the Irish private car ODEX when both proposed changes of using MJ/km as the unit consumption measure and modeling the stock by vintage were applied. The overall effect of the revised transport ODEX calculation does not show a significant increase in energy savings associated with the value of the ODEX indicator (0.82%). However the purpose was to improve the methodology of how the ODEX was being calculated, not necessarily increasing the savings

The impact of the invasive alien plant, impatiens glandulifera, on pollen transfer networks

Biological invasions are a threat to the maintenance of ecological processes, including pollination. Plant-flower visitor networks are traditionally used as a surrogated for pollination at the community level, despite they do not represent the pollination process, which takes place at the stigma of plants where pollen grains are deposited. Here we investigated whether the invasion of the alien plant Impatiens glandulifera (Balsaminaceae) affects pollen transfer at the community level. We asked whether more alien pollen is deposited on the stigmas of plants on invaded sites, whether deposition is affected by stigma type (dry, semidry and wet) and whether the invasion of I. glandulifera changes the structure of the resulting pollen transfer networks. We sampled stigmas of plants on 10 sites invaded by I. glandulifera (hereafter, balsam) and 10 non-invaded control sites. All 20 networks had interactions with balsam pollen, although significantly more balsam pollen was found on plants with dry stigmas in invaded areas. Balsam pollen deposition was restricted to a small subset of plant species, which is surprising because pollinators are known to carry high loads of balsam pollen. Balsam invasion did not affect the loading of native pollen, nor did it affect pollen transfer network properties; networks were modular and poorly nested, both of which are likely to be related to the specificity of pollen transfer interactions. Our results indicate that pollination networks become more specialized when moving from the flower visitation to the level of pollen transfer networks. Therefore, caution is needed when inferring pollination from patterns of insect visitation or insect pollen loads as the relationship between these and pollen deposition is not straightforward. © 2015 Emer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Amphotericin B enhances the synthesis and release of the immunosuppressive agent gliotoxin from the pulmonary pathogen Aspergillus fumigatus

Exposure of the pulmonary pathogen Aspergillus fumigatus to amphotericin B alters membrane permeability as indicated by the escape of amino acids and protein from the mycelium. Amphotericin B exposure for periods of 2-4 h also leads to increased release of the immunosuppressive agent gliotoxin into the surrounding culture medium. Examination of the intracellular gliotoxin concentration following exposure to amphotericin B indicated elevated levels within the hyphae as well as in the culture medium - an effect which was also evident upon exposure of A. fumigatus to DMSO. These results indicate that in parallel with the ability of amphotericinBto act as a fungistatic agent it can also induce the synthesis of gliotoxin and facilitate its release by increasing the permeability of the fungal cell membrane. Increased synthesis of gliotoxin may result from the commencement of secondary metabolism in the presence of amphotericin B. The ability of amphotericin B to enhance the synthesis and release of gliotoxin may exacerbate the effects of the toxin and facilitate fungal invasion of pulmonary tissue

Translocation of proteins homologous to human neutrophil p47phox and p67phox to the cell membrane in activated hemocytes of Galleria mellonella

Activation of the superoxide forming respiratory burst oxidase of human neutrophils, crucial in host defence, requires the cytosolic proteins p47phox and p67phox which translocate to the plasma membrane upon cell stimulation and activate flavocytochrome b558, the redox centre of this enzyme system. We have previously demonstrated the presence of proteins (67 and 47 kDa) in hemocytes of the insect Galleria mellonella homologous to proteins of the superoxide-forming NADPH oxidase complex of neutrophils. The work presented here illustrates for the first time translocation of homologous hemocyte proteins, 67 and 47 kDa from the cytosol to the plasma membrane upon phorbol 12-myristate 13 acetate (PMA)activation. In hemocytes, gliotoxin (GT), the fungal secondary metabolite significantly suppressed PMA-induced superoxide generation in a concentration dependent manner and reduced translocation to basel nonstimulated levels. Primarily these results correlate translocation of hemocyte 47 and 67 kDa proteins with PMA induced oxidase activity. Collectively results presented here, demonstrate further cellular and functional similarities between phagocytes of insects and mammals and further justify the use of insects in place of mammals for modelling the innate immune response to microbial pathogens