A Quantitative Assessment of the Role of the Parasite Amoebophrya in the Termination of Alexandrium fundyense Blooms within a Small Coastal Embayment

Parasitic dinoflagellates of the genus Amoebophrya infect free-living dinoflagellates, some of which can cause harmful algal blooms (HABs). High prevalence of Amoebophrya spp. has been linked to the decline of some HABs in marine systems. The objective of this study was to evaluate the impact of Amoebophrya spp. on the dynamics of dinoflagellate blooms in Salt Pond (MA, USA), particularly the harmful species Alexandrium fundyense. The abundance of Amoebophrya life stages was estimated 3–7 days per week through the full duration of an annual A. fundyense bloom using fluorescence in situ hybridization coupled with tyramide signal amplification (FISH- TSA). More than 20 potential hosts were recorded including Dinophysis spp., Protoperidinium spp. and Gonyaulax spp., but the only dinoflagellate cells infected by Amoebophrya spp. during the sampling period were A. fundyense. Maximum A. fundyense concentration co-occurred with an increase of infected hosts, followed by a massive release of Amoebophrya dinospores in the water column. On average, Amoebophrya spp. infected and killed ∼30% of the A. fundyense population per day in the end phase of the bloom. The decline of the host A. fundyense population coincided with a dramatic life-cycle transition from vegetative division to sexual fusion. This transition occurred after maximum infected host concentrations and before peak infection percentages were observed, suggesting that most A. fundyense escaped parasite infection through sexual fusion. The results of this work highlight the importance of high frequency sampling of both parasite and host populations to accurately assess the impact of parasites on natural plankton assemblages.     

Effective Treatment of Respiratory Alphaherpesvirus Infection Using RNA Interference

Background

Equine herpesvirus type 1 (EHV-1), a member of the Alphaherpesvirinae, is spread via nasal secretions and causes respiratory disease, neurological disorders and abortions. The virus is a significant equine pathogen, but current EHV-1 vaccines are only partially protective and effective metaphylactic and therapeutic agents are not available. Small interfering RNAs (siRNA''s), delivered intranasally, could prove a valuable alternative for infection control. siRNA''s against two essential EHV-1 genes, encoding the viral helicase (Ori) and glycoprotein B, were evaluated for their potential to decrease EHV-1 infection in a mouse model.

Methodology/Principal Fndings

siRNA therapy in vitro significantly reduced virus production and plaque size. Viral titers were reduced 80-fold with 37.5 pmol of a single siRNA or with as little as 6.25 pmol of each siRNA when used in combination. siRNA therapy in vivo significantly reduced viral replication and clinical signs. Intranasal treatment did not require a transport vehicle and proved effective when given up to 12 h before or after infection.

Conclusions/Significance

siRNA treatment has potential for both prevention and early treatment of EHV-1 infections.     

Kinetic studies of iron deposition in horse spleen ferritin using O2 as oxidant

An optical flow cell provided a means to conveniently measure the rate of successive Fe(2+) oxidation reactions catalyzed by horse spleen ferritin (HoSF) to determine if both ferroxidase and mineral core Fe(2+) oxidation reactions occur. The oxygen concentration and pH were held constant and multiple additions of Fe(2+)/HoSF ratios of 1, 10, 100, 150, 250 and 400 were conducted, creating core sizes ranging from 12 to 2800. During these oxidations, the absence of nonspecific Fe(OH)(3) formation and the presence (>95%) of Fe(OH)(3) deposited within the core of HoSF demonstrated the validity of monitoring iron deposition into HoSF by this procedure. Initial rates for oxidation of 5-50 Fe(2+)/HoSF established that the reaction is overall first order in Fe(2+) concentration. However, when full progress curves were analyzed at a variety of Fe(2+)/HoSF ratios, two first-order reactions (k(1) approximately 0.035 s(-1) and k(2) approximately 0.007 s(-1)) were found to contribute to the overall Fe(2+) oxidation reaction. The proportion of the fast reaction increased with increasing Fe(2+)/HoSF ratio until at approximately 400, it was the dominant reaction. For the Fe(2+)/HoSF ratios examined, the overall rate of iron deposition is independent of the size of the mineral core, a result suggesting that an increasing mineral core size does not enhance the rate of Fe(2+) oxidation. Comparison of successive additions of 1.0 Fe(2+)/HoSF showed that oxidation of the first 8-10 Fe(2+) produced a Fe(III) species with a lower molar absorptivity per Fe(III) than that of the bulk core. Measurement of the H(+)/Fe(2+) ratio confirmed this difference in behavior by giving an H(+)/Fe(2+) ratio of approximately 1.0 below and 2.0 for ratios >30 Fe(2+)/HoSF. The faster reaction was attributed to ferroxidase catalysis and the slow reaction to nonspecific ferroxidase activity of the HoSF protein shell.     

Gram-positive bacterial superantigen outside-in signaling causes toxic shock syndrome

Staphylococcus aureus and Streptococcus pyogenes (group A streptococci) are Gram-positive pathogens capable of producing a variety of bacterial exotoxins known as superantigens. Superantigens interact with antigen-presenting cells (APCs) and T cells to induce T cell proliferation and massive cytokine production, which leads to fever, rash, capillary leak and subsequent hypotension, the major symptoms of toxic shock syndrome. Both S. aureus and group A streptococci colonize mucosal surfaces, including the anterior nares and vagina for S. aureus, and the oropharynx and less commonly the vagina for group A streptococci. However, due to their abilities to secrete a variety of virulence factors, the organisms can also cause illnesses from the mucosa. This review provides an updated discussion of the biochemical and structural features of one group of secreted virulence factors, the staphylococcal and group A streptococcal superantigens, and their abilities to cause toxic shock syndrome from a mucosal surface. The main focus of this review, however, is the abilities of superantigens to induce cytokines and chemokines from epithelial cells, which has been linked to a dodecapeptide region that is relatively conserved among all superantigens and is distinct from the binding sites required for interactions with APCs and T cells. This phenomenon, termed outside-in signaling, acts to recruit adaptive immune cells to the submucosa, where the superantigens can then interact with those cells to initiate the final cytokine cascades that lead to toxic shock syndrome.     

Enterococcus faecalis Inhibits Superantigen Toxic Shock Syndrome Toxin-1-Induced Interleukin-8 from Human Vaginal Epithelial Cells through Tetramic Acids

The vaginal mucosa can be colonized by many bacteria including commensal organisms and potential pathogens, such as Staphylococcus aureus. Some strains of S. aureus produce the superantigen toxic shock syndrome toxin-1, which can penetrate the vaginal epithelium to cause toxic shock syndrome. We have observed that a female was mono-colonized with Enterococcus faecalis vaginally as tested in aerobic culture, even upon repeated culture for six months, suggesting this organism was negatively influencing colonization by other bacteria. In recent studies, we demonstrated an “outside-in” mechanism of cytokine signaling and consequent inflammation that facilitates the ability of potential pathogens to initiate infection from mucosal surfaces. Thus, we hypothesized that this strain of E. faecalis may make anti-inflammatory factors which block disease progression of more pathogenic organisms. E. faecalis MN1 inhibited interleukin-8 production from human vaginal epithelial cells in response to the vaginal pathogens Candida albicans, Gardnerella vaginalis, and Neisseria gonorrhoeae, as well as to toxic shock syndrome toxin-1. We further demonstrated that this organism secretes two tetramic acid compounds which appear responsible for inhibition of interleukin-8 production, as well as inhibition of T cell proliferation due to toxic shock syndrome toxin-1. Microbicides that include anti-inflammatory molecules, such as these tetramic acid compounds naturally produced by E. faecalis MN1, may be useful in prevention of diseases that develop from vaginal infections.     

Spatial indicators for nature conservation from European to local scale

The paper presents an overview of the objectives and exemplary results of the FP 5 project “Spatial Indicators for European Nature Conservation” (SPIN). The SPIN project is focused on the development and testing of advanced classification methods and spatial indicators based on multisensor satellite data and GIS to accomplish monitoring and management tasks in the context of Natura 2000 and nature conservation. A representative selection of eight regional test areas covers a pan-European network and allows comparative investigations to provide accepted recommendations for regional and European nature conservation. The selected results of four case studies are presented and discussed. The range of work covers the production of regional and local habitat maps by object-oriented classification, a case-based reasoning method for change detection as a management support tool for planning and regulating local land use, the selection and application of structural indicators for the monitoring of Natura 2000 habitats and the downscaling and disaggregation of soil information. Results and the further implementation of presented methods are discussed in the conclusions.     

Non-reductive iron release from horse spleen ferritin using desferoxamine chelation

The rate of Fe³⁺ release from horse spleen ferritin (HoSF) was measured using the Fe³⁺-specific chelator desferoxamine (DES). The reaction consists of two kinetic phases. The first is a rapid non-linear reaction followed by a slower linear reaction. The overall two-phase reaction was resolved into three kinetic events: 1) a rapid first-order reaction in HoSF (k₁); 2) a second slower first-order reaction in HoSF (k₂); and 3) a zero-order slow reaction in HoSF (k₃). The zero-order reaction was independent of DES concentration. The two first-order reactions had a near zero-order dependence on DES concentration and were independent of pH from 6.8 to 8.2. The two first-order reactions accounted for 6-9 rapidly reacting Fe³⁺ ions. Activation energies of 10.5 ± 0.8, 13.5 ± 2.0 and 62.4 ± 2.1 kJ/mol were calculated for the kinetic events associated with k₁, k₂, and k₃, respectively. Iron release occurs by: 1) a slow zero-order rate-limiting reaction governed by k₃ and corresponding to the dissociation of Fe³⁺ ions from the FeOOH core that bind to an Fe³⁺ binding site designated as site 1 (proposed to be within the 3-fold channel); 2) transfer of Fe³⁺ from site 1 to site 2 (a second binding site in the 3-fold channel) (k₂); and 3) rapid iron loss from site 2 to DES (k₁).     

Discrimination between viable and dead Xanthomonas fragariae in strawberry using viability PCR

Xanthomonas fragariae is the causal agent of an important bacterial disease in strawberry production regions worldwide and a quarantine plant pathogen in many countries including New Zealand. Xanthomonas fragariae mainly infects the foliage of strawberry plants but can also infect the calyx tissue associated with strawberry fruit. Fresh strawberries are a high-value internationally traded commodity that has a short shelf-life. When making biosecurity decisions based on the finding of a quarantine organism such as X. fragariae by PCR, one of the major challenges is the inability to differentiate positive results originating from viable or dead cells. Viability PCR (vPCR) is a technique that selectively inhibits PCR amplification of DNA derived from dead cells through the use of a nucleic acid intercalating dye, for example, PEMAX™. A vPCR protocol has been optimized to enable rapid detection of viable X. fragariae in a tissue sample. PEMAX™ treatment resulted in complete inhibition of PCR amplification of 10⁸–10³ cfu/ml dead X. fragariae cells in strawberry host tissue. The most important parameters for optimization were the dilution of the sample, amplicon length and choice of nucleic acid intercalating dye. This study provides a rapid protocol to discriminate between viable and dead X. fragariae in strawberry in a phytosanitary environment. This test will help timely decisions to be made at the border on imported fresh strawberry consignments that test positive for X. fragariae.     

IL-22 is expressed by the invasive trophoblast of the equine (Equus caballus) chorionic girdle

The invasive trophoblast cells of the equine placenta migrate into the endometrium to form endometrial cups, dense accumulations of trophoblast cells that produce equine chorionic gonadotropin between days 40 and 120 of normal pregnancy. The mechanisms by which the trophoblast cells invade the endometrium while evading maternal immune destruction are poorly defined. A gene expression microarray analysis performed on placental tissues obtained at day 34 of gestation revealed a >900-fold upregulation of mRNA encoding the cytokine IL-22 in chorionic girdle relative to noninvasive chorion. Quantitative RT-PCR assays were used to verify high expression of IL-22 in chorionic girdle. Additional quantitative RT-PCR analysis showed a striking increase in IL-22 mRNA expression in chorionic girdle from days 32 to 35 and an absence of IL-22 expression in other conceptus tissues. Bioinformatic analysis and cDNA sequencing confirmed the predicted length of horse IL-22, which carries a 3' extension absent in IL-22 genes of humans and mice, but present in the cow and pig. Our discovery of IL-22 in the chorionic girdle is a novel finding, as this cytokine has been previously reported in immune cells only. IL-22 has immunoregulatory functions, with primary action on epithelial cells. mRNA of IL-22R1 was detected in pregnant endometrium at levels similar to other equine epithelia. Based upon these findings, we hypothesize that IL-22 cytokine produced by the chorionic girdle binds IL-22R1 on endometrium, serving as a mechanism of fetal-maternal communication by modulating endometrial responses to trophoblast invasion.