Detection of dsRNA from Cleistothecia and Conidia of the Grape Powdery Mildew Pathogen, Uncinula Necator

Double-stranded RNA species ranging in molecular weight from 0.95 to 6.3 × 106 were detected in grapevines in New York. We recently showed that two of the species (Mr = 5.3 and 4.4 × 106) are associated with rupestris stem pitting disease. In this report, we show that the other eight detectable dsRNA species are associated with the powdery mildew fungus, Uncinula necator. These dsRNAs associated with the powdery mildew fungus were previously detected in leaves and epidermal stem tissue of grapevines infected with powdery mildew. The same dsRNA species were also detected from extracts of isolated cleistothecia and conidia of U. necator devoid of plant tissue. Isometric and rigid rodlike particles were observed in single cleistothecia preparations when examined under transmission electron microscopy.

Cultivar-specific effects of pathogen testing on storage root yield of sweet potato, Ipomoea batatas (L.) Lam.

The accumulation and perpetuation of viral pathogens over generations of clonal propagation in crop species such as sweet potato, Ipomoea batatas,inevitably result in a reduction in crop yield and quality. This study was conducted at Bundaberg, Australia to compare the productivity of field-derived and pathogen-tested (PT)clones of 14 sweet potato cultivars and the yield benefits of using healthy planting materials. The field-derived clonal materials were exposed to the endemic viruses, while the PT clones were subjected to thermotherapy and meristem-tip culture to eliminate viral pathogens. The plants were indexed for viruses using nitrocellulose membrane-enzyme-linked immunosorbent assay and graft-inoculations onto Ipomoea setosa. A net benefit of 38% in storage root yield was realised from using PT materials in this study.Conversely, in a similar study previously conducted at Kerevat, Papua New Guinea (PNG), a net deficit of 36% was realised. This reinforced our finding that the response to pathogen testing was cultivar dependent and that the PNG cultivars in these studies generally exhibited increased tolerance to the endemic viruses present at the respective trial sites as manifested in their lack of response from the use of PT clones. They may be useful sources for future resistance breeding efforts. Nonetheless, the potential economic gain from using PT stocks necessitates the use of pathogen testing on virus-susceptible commercial cultivars.

Investigation into the introduction of clonal strains of Pseudomonas aeruginosa to the cystic fibrosis population by consumption of raw salad vegetables

Most salad vegetables are eaten fresh by consumers. However, raw vegetables may pose a risk of transmitting opportunistic bacteria to immunocompromised people, including cystic fibrosis (CF) patients. In particular, CF patients are vulnerable to chronic Pseudomonas aeruginosa lung infections and this organism is the primary cause of morbidity and mortality in this group. Clonal variants of P. aeruginosa have been identified as emerging threats to people afflicted with CF; however it has not yet been proven from where these clones originate or how they are transmitted. Due to the organisms‟ aquatic environmental niche, it was hypothesised that vegetables may be a source of these clones. To test this hypothesis, lettuce, tomatoes, mushrooms and bean sprout packages (n = 150) were analysed from a green grocer, supermarket and farmers‟ market within the Brisbane region, availability permitting. The internal and external surfaces of the vegetables were separately analysed for the presence of clonal strains of P. aeruginosa using washings and homogenisation techniques, respectively. This separation was in an attempt to establish which surface was contaminated, so that recommendations could be made to decrease or eliminate P. aeruginosa from these foods prior to consumption. Soil and water samples (n = 17) from local farms were also analysed for the presence of P. aeruginosa. Presumptive identification of isolates recovered from these environmental samples was made based on growth on Cetrimide agar at 42°C, presence of the cytochrome-oxidase enzyme and inability to ferment lactose. P. aeruginosa duplex real-time polymerase chain reaction assay (PAduplex) was performed on all bacterial isolates presumptively identified as P. aeruginosa. Enterobacterial repetitive intergenic consensus strain typing PCR (ERIC-PCR) was subsequently performed on confirmed bacterial isolates. Although 72 P. aeruginosa were isolated, none of these proved to be clonal strains. The significance of these findings is that vegetables may pose a risk of transmitting sporadic strains of P. aeruginosa to people afflicted with CF and possibly, other immunocompromised people.

Development of an autonomous unmanned aerial system to collect time-stamped samples from the atmosphere and localize potential pathogen sources

This paper presents the hardware development and testing of a new concept for air sampling via the integration of a prototype spore trap onboard an unmanned aerial system (UAS).We propose the integration of a prototype spore trap onboard a UAS to allow multiple capture of spores of pathogens in single remote locations at high or low altitude, otherwise not possible with stationary sampling devices.We also demonstrate the capability of this system for the capture of multiple time-stamped samples during a single mission.Wind tunnel testing was followed by simulation, and flight testing was conducted to measure and quantify the spread during simulated airborne air sampling operations. During autonomous operations, the onboard autopilot commands the servo to rotate the sampling device to a new indexed location once the UAS vehicle reaches the predefined waypoint or set of waypoints (which represents the region of interest). Time-stamped UAS data are continuously logged during the flight to assist with analysis of the particles collected. Testing and validation of the autopilot and spore trap integration, functionality, and performance is described. These tools may enhance the ability to detect new incursions of spores

The oral health of critically ill children : an observational cohort study

Aims and objectives: This study will describe the oral health status of critically ill children over time spent in the paediatric intensive care unit, examine influences on the development of poor oral health and explore the relationship between dysfunctional oral health and healthcare-associated infections. Background: The treatment modalities used to support children experiencing critical illness and the progression of critical illness may result in dysfunction in the oral cavity. In adults, oral health has been shown to worsen during critical illness as well as influence systemic health. Design: A prospective observational cohort design was used. Method: The study was undertaken at a single tertiary-referral Paediatric Intensive Care Unit. Oral health status was measured using the Oral Assessment Scale and culturing oropharyngeal flora. Information was also collected surrounding the use of supportive therapies, clinical characteristics of the children and the occurrence of healthcare-associated infections. Results: Of the 46 participants, 63% (n = 32) had oral dysfunction and 41% (n = 19) demonstrated pathogenic oropharyngeal colonisation during their critical illness. The potential systemic pathogens isolated from the oropharynx and included Candida sp., Staphylococcus aureus, Haemophilus influenzae, Enterococcus sp. and Pseudomonas aeruginosa. The severity of critical illness had a significant positive relationship (p < 0·05) with pathogenic and absent colonisation of the oropharynx. Sixty-three percent of healthcare-associated infections involved the preceding or simultaneous colonisation of the oropharynx by the causative pathogen. Conclusions: This study suggests paediatric oral health to be frequently dysfunctional and the oropharynx to repeatedly harbour potential systemic pathogens during childhood critical illness. Relevance to clinical practice: Given the frequency of poor oral health during childhood critical illness in this study and the subsequent potential systemic consequences, evidence based oral hygiene practices should be developed and validated to guide clinicians when nursing critically ill children.

Recombination, decreased host specificity and increased mobility may have driven the emergence of maize streak virus as an agricultural pathogen

Maize streak virus (MSV; family Geminiviridae, genus Mastrevirus), the causal agent of maize streak disease, ranks amongst the most serious biological threats to food security in subSaharan Africa. Although five distinct MSV strains have been currently described, only one of these - MSV-A - causes severe disease in maize. Due primarily to their not being an obvious threat to agriculture, very little is known about the 'grass-adapted' MSV strains, MSV-B, -C, -D and -E. Since comparing the genetic diversities, geographical distributions and natural host ranges of MSV-A with the other MSV strains could provide valuable information on the epidemiology, evolution and emergence of MSV-A, we carried out a phylogeographical analysis of MSVs found in uncultivated indigenous African grasses. Amongst the 83 new MSV genomes presented here, we report the discovery of six new MSV strains (MSV-F to -K). The non-random recombination breakpoint distributions detectable with these and other available mastrevirus sequences partially mirror those seen in begomoviruses, implying that the forces shaping these breakpoint patterns have been largely conserved since the earliest geminivirus ancestors. We present evidence that the ancestor of all MSV-A variants was the recombinant progeny of ancestral MSV-B and MSV-G/-F variants. While it remains unknown whether recombination influenced the emergence of MSV-A in maize, our discovery that MSV-A variants may both move between and become established in different regions of Africa with greater ease, and infect more grass species than other MSV strains, goes some way towards explaining why MSV-A is such a successful maize pathogen. © 2008 SGM.

Maize streak virus: An old and complex 'emerging' pathogen

Maize streak virus (MSV; Genus Mastrevirus, Family Geminiviridae) occurs throughout Africa, where it causes what is probably the most serious viral crop disease on the continent. It is obligately transmitted by as many as six leafhopper species in the Genus Cicadulina, but mainly by C. mbila Naudé and C. storeyi. In addition to maize, it can infect over 80 other species in the Family Poaceae. Whereas 11 strains of MSV are currently known, only the MSV-A strain is known to cause economically significant streak disease in maize. Severe maize streak disease (MSD) manifests as pronounced, continuous parallel chlorotic streaks on leaves, with severe stunting of the affected plant and, usuallly, a failure to produce complete cobs or seed. Natural resistance to MSV in maize, and/or maize infections caused by non-maize-adapted MSV strains, can result in narrow, interrupted streaks and no obvious yield losses. MSV epidemiology is primarily governed by environmental influences on its vector species, resulting in erratic epidemics every 3-10 years. Even in epidemic years, disease incidences can vary from a few infected plants per field, with little associated yield loss, to 100% infection rates and complete yield loss. Taxonomy: The only virus species known to cause MSD is MSV, the type member of the Genus Mastrevirus in the Family Geminiviridae. In addition to the MSV-A strain, which causes the most severe form of streak disease in maize, 10 other MSV strains (MSV-B to MSV-K) are known to infect barley, wheat, oats, rye, sugarcane, millet and many wild, mostly annual, grass species. Seven other mastrevirus species, many with host and geographical ranges partially overlapping those of MSV, appear to infect primarily perennial grasses. Physical properties: MSV and all related grass mastreviruses have single-component, circular, single-stranded DNA genomes of approximately 2700 bases, encapsidated in 22 × 38-nm geminate particles comprising two incomplete T = 1 icosahedra, with 22 pentameric capsomers composed of a single 32-kDa capsid protein. Particles are generally stable in buffers of pH 4-8. Disease symptoms: In infected maize plants, streak disease initially manifests as minute, pale, circular spots on the lowest exposed portion of the youngest leaves. The only leaves that develop symptoms are those formed after infection, with older leaves remaining healthy. As the disease progresses, newer leaves emerge containing streaks up to several millimetres in length along the leaf veins, with primary veins being less affected than secondary or tertiary veins. The streaks are often fused laterally, appearing as narrow, broken, chlorotic stripes, which may extend over the entire length of severely affected leaves. Lesion colour generally varies from white to yellow, with some virus strains causing red pigmentation on maize leaves and abnormal shoot and flower bunching in grasses. Reduced photosynthesis and increased respiration usually lead to a reduction in leaf length and plant height; thus, maize plants infected at an early stage become severely stunted, producing undersized, misshapen cobs or giving no yield at all. Yield loss in susceptible maize is directly related to the time of infection: Infected seedlings produce no yield or are killed, whereas plants infected at later times are proportionately less affected. Disease control: Disease avoidance can be practised by only planting maize during the early season when viral inoculum loads are lowest. Leafhopper vectors can also be controlled with insecticides such as carbofuran. However, the development and use of streak-resistant cultivars is probably the most effective and economically viable means of preventing streak epidemics. Naturally occurring tolerance to MSV (meaning that, although plants become systemically infected, they do not suffer serious yield losses) has been found, which has primarily been attributed to a single gene, msv-1. However, other MSV resistance genes also exist and improved resistance has been achieved by concentrating these within individual maiz genotypes. Whereas true MSV immunity (meaning that plants cannot be symptomatically infected by the virus) has been achieved in lines that include multiple small-effect resistance genes together with msv-1, it has proven difficult to transfer this immunity into commercial maize genotypes. An alternative resistance strategy using genetic engineering is currently being investigated in South Africa. Useful websites: 〈http://www.mcb.uct.ac.za/MSV/mastrevirus.htm〉; 〈http://www. danforthcenter.org/iltab/geminiviridae/geminiaccess/mastrevirus/Mastrevirus. htm〉. © 2009 Blackwell Publishing Ltd.

Chlamydia pneumoniae : modern insights into an ancient pathogen

Chlamydia pneumoniae is an enigmatic human and animal pathogen. Originally discovered in association with acute human respiratory disease, it is now associated with a remarkably wide range of chronic diseases as well as having a cosmopolitan distribution within the animal kingdom. Molecular typing studies suggest that animal strains are ancestral to human strains and that C. pneumoniae crossed from animals to humans as the result of at least one relatively recent zoonotic event. Whole genome analyses appear to support this concept – the human strains are highly conserved whereas the single animal strain that has been fully sequenced has a larger genome with several notable differences. When compared to the other, better known chlamydial species that is implicated in human infection, Chlamydia trachomatis, C. pneumoniae demonstrates pertinent differences in its cell biology, development, and genome structure. Here, we examine the characteristic facets of C. pneumoniae biology, offering insights into the diversity and evolution of this silent and ancient pathogen.

Hyperlipidemia impaired innate immune response to periodontal pathogen Porphyromonas gingivalis in apolipoprotein E knockout mice

A finely-tuned innate immune response plays a pivotal role in protecting host against bacterial invasion during periodontal disease progression. Hyperlipidemia has been suggested to exacerbate periodontal health condition. However, the underlying mechanism has not been addressed. In the present study, we investigated the effect of hyperlipidemia on innate immune responses to periodontal pathogen Porphyromonas gingivalis infection. Apolipoprotein E-deficient and wild-type mice at the age of 20 weeks were used for the study. Peritoneal macrophages were isolated and subsequently used for the study of viable P. gingivalis infection. ApoE−/− mice demonstrated inhibited iNOS production and impaired clearance of P. gingivalis in vitro and in vivo; furthermore, ApoE−/− mice displayed disrupted cytokine production pattern in response to P. gingivalis, with a decreased production of tumor necrosis factor-α, interleukin-6 (IL-6), IL-1β and monocyte chemotactic protein-1. Microarray data demonstrated that Toll-like receptor (TLR) and NOD-like receptor (NLR) pathway were altered in ApoE−/− mice macrophages; further analysis of pattern recognition receptors (PRRs) demonstrated that expression of triggering receptors on myeloid cells-1 (TREM-1), an amplifier of the TLR and NLR pathway, was decreased in ApoE−/− mice macrophages, leading to decreased recruitment of NF-κB onto the promoters of the TNF-α and IL-6. Our data suggest that in ApoE−/− mice hyperlipidemia disrupts the expression of PRRs, and cripples the host’s capability to generate sufficient innate immune response to P. gingivalis, which may facilitate immune evasion, subgingival colonization and establishment of P. gingivalis in the periodontal niche.

A systematic review of mathematical models of mosquito-borne pathogen transmission: 1970-2010

Mathematical models of mosquito-borne pathogen transmission originated in the early twentieth century to provide insights into how to most effectively combat malaria. The foundations of the Ross–Macdonald theory were established by 1970. Since then, there has been a growing interest in reducing the public health burden of mosquito-borne pathogens and an expanding use of models to guide their control. To assess how theory has changed to confront evolving public health challenges, we compiled a bibliography of 325 publications from 1970 through 2010 that included at least one mathematical model of mosquito-borne pathogen transmission and then used a 79-part questionnaire to classify each of 388 associated models according to its biological assumptions. As a composite measure to interpret the multidimensional results of our survey, we assigned a numerical value to each model that measured its similarity to 15 core assumptions of the Ross–Macdonald model. Although the analysis illustrated a growing acknowledgement of geographical, ecological and epidemiological complexities in modelling transmission, most models during the past 40 years closely resemble the Ross–Macdonald model. Modern theory would benefit from an expansion around the concepts of heterogeneous mosquito biting, poorly mixed mosquito-host encounters, spatial heterogeneity and temporal variation in the transmission process.

The effect of nitroxides on swarming motility and biofilms, multicellular behaviors in Pseudomonas aeruginosa

The ability of NO to induce biofilm dispersion has been well established. Here we investigated the effect of nitroxides (sterically hindered nitric oxide analogues) on biofilm formation and swarming motility in Pseudomonas aeruginosa. A transposon mutant unable to produce nitric oxide endogenously (nirS) was deficient in swarming motility relative to wild type and the complemented strain. Moreover, expression of the nirS gene was up-regulated by 9.65-fold in wild type swarming cells when compared to planktonic cells. Wild type swarming levels were substantially restored upon exogenous addition of nitroxide containing compounds, consistent with the hypothesis that NO is necessary for swarming motility. Here, we showed that nitroxides not only mimicked the dispersal activity of NO, but also prevented biofilms from forming in flow cell chambers. In addition, a nirS transposon mutant was deficient in biofilm formation relative to wild type and the complemented strain, thus implicating NO in the formation of biofilms. Intriguingly despite its stand alone action in inhibiting biofilm formation and promoting dispersal, a nitroxide partially restored the ability of a nirS mutant to form biofilms.

Recasting the theory of mosquito-borne pathogen transmission dynamics and control

Mosquito-borne diseases pose some of the greatest challenges in public health, especially in tropical and sub-tropical regions of theworld. Efforts to control these diseases have been underpinned by a theoretical framework developed for malaria by Ross and Macdonald, including models, metrics for measuring transmission, and theory of control that identifies key vulnerabilities in the transmission cycle. That framework, especially Macdonald’s formula for R0 and its entomological derivative, vectorial capacity, are nowused to study dynamics and design interventions for many mosquito-borne diseases. A systematic review of 388 models published between 1970 and 2010 found that the vast majority adopted the Ross–Macdonald assumption of homogeneous transmission in a well-mixed population. Studies comparing models and data question these assumptions and point to the capacity to model heterogeneous, focal transmission as the most important but relatively unexplored component in current theory. Fine-scale heterogeneity causes transmission dynamics to be nonlinear, and poses problems for modeling, epidemiology and measurement. Novel mathematical approaches show how heterogeneity arises from the biology and the landscape on which the processes of mosquito biting and pathogen transmission unfold. Emerging theory focuses attention on the ecological and social context formosquito blood feeding, themovement of both hosts and mosquitoes, and the relevant spatial scales for measuring transmission and for modeling dynamics and control.

Nanohybrids of silver particles on clay platelets delaminate Pseudomonas biofilms

Aim To evaluate the effectiveness of novel nanohybrids, composed of silver nanoparticles and nanoscale silicate platelets, to clear Pseudomonas aeruginosa biofilms. Materials & methods The nanohybrids were manufactured from an in situ reduction of silver salts in the silicate platelet dispersion, and then applied to biofilms in vitro and in vivo. Results In reference to the biocidal effects of gentamycin, the nanohybrids mitigated the spreading of the biofilms, and initiated robust cell death and exfoliation from the superficial layers of the biofilms in vitro. In vivo, the nanohybrids exhibited significant therapeutic effects by eliminating established biofilms from infected corneas and promoting the recovery of corneal integrity. Conclusion All of the evaluations indicate the high potency of the newly developed silver nanoparticle/nanoscale silicate platelet nanohybrids for eliminating biofilms.

Cloning, expression, characterisation and mutational analysis of l-aspartate oxidase from Pseudomonas putida

L-Amino acid oxidases (LAAOs) are useful catalysts for the deracemisation of racemic amino acid sub-strates when combined with abiotic reductants. The gene nadB encoding the L-aspartate amino acid oxidase from Pseudomonas putida (PpLASPO) has been cloned and expressed in E. coli. The purified PpLASPO enzyme displayed a K M for l-aspartic acid of 2.26 mM and a k cat = 10.6 s −1 , with lower activity also displayed towards L-asparagine, for which pronounced substrate inhibition was also observed. The pH optimum of the enzyme was recorded at pH 7.4. The enzyme was stable for 60 min at up to 40 • C, but rapid losses in activity were observed at 50 • C. A mutational analysis of the enzyme, based on its sequence homology with the LASPO from E. coli of known structure, appeared to confirm roles in substrate binding or catalysis for residues His244, His351, Arg386 and Arg290 and also for Thr259 and Gln242. The high activity of the enzyme, and its promiscuous acceptance of both L-asparagine and L-glutamate as substrates, if with low activity, suggests that PpLASPO may provide a good model enzyme for evolution studies towards AAOs of altered or improved properties in the future.