Metabolomic Tools for Secondary Metabolite Discovery from Marine Microbial Symbionts

Marine invertebrate-associated symbiotic bacteria produce a plethora of novel secondary metabolites which may be structurally unique with interesting pharmacological properties. Selection of strains usually relies on literature searching, genetic screening and bioactivity results, often without considering the chemical novelty and abundance of secondary metabolites being produced by the microorganism until the time-consuming bioassay-guided isolation stages. To fast track the selection process, metabolomic tools were used to aid strain selection by investigating differences in the chemical profiles of 77 bacterial extracts isolated from cold water marine invertebrates from Orkney, Scotland using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR) spectroscopy. Following mass spectrometric analysis and dereplication using an Excel macro developed in-house, principal component analysis (PCA) was employed to differentiate the bacterial strains based on their chemical profiles. NMR ¹H and correlation spectroscopy (COSY) were also employed to obtain a chemical fingerprint of each bacterial strain and to confirm the presence of functional groups and spin systems. These results were then combined with taxonomic identification and bioassay screening data to identify three bacterial strains, namely Bacillus sp. 4117, Rhodococcus sp. ZS402 and Vibrio splendidus strain LGP32, to prioritize for scale-up based on their chemically interesting secondary metabolomes, established through dereplication and interesting bioactivities, determined from bioassay screening.     

Expert opinion based modelling of the risk of human infection with H5N1 through the consumption of poultry meat in Germany

According to current scientific opinion the risk of human infection with H5N1 via preparation and consumption of poultry meat is negligible.This opinion has not yet been challenged by a formal risk assessment, due to the lack of empirical data. We have developed a scenario pathway model as a conceptual framework for a formal assessment of the H5N1 risk to humans through consumption of poultry meat and parameterise the model using information derived from expert opinions. The aim of this study was to investigate whether the notion of an overall negligible risk via the oral infection route is consistent with ad hoc data and expert opinions on the relevant parameters of the model.The model is mainly based on expert opinion. A stochastic Monte-Carlo simulation was conducted which took into consideration (amongst others) the exposure and infection of chicken (broiler and layer), turkeys, ducks and geese, the probabilities of detection prior to slaughter, virus survival and contamination during slaughter, as well as during the cutting and preparation of meat in commercial plants and in private households, respectively. The empirical consumption pattern for poultry meat in Germany was taken into account in the simulation.The results show that the risk for the individual consumer is practically zero whereas up to 23 cases per year in Germany might occur if the upper (more pessimistic) ranges of the expert opinions apply.The finding of a low but non-negligible risk to the population is discussed in relation to the epidemiological information available from recent outbreaks in South East Asia.     

Methicillin‐resistant Staphylococcus aureus in urban Norway rat (Rattus norvegicus) populations: Epidemiology and the impacts of kill‐trapping

Urban Norway rat (Rattus norvegicus) populations can carry the bacteria methicillin‐resistant Staphylococcus aureus (MRSA). There are numerous knowledge gaps in the epidemiology of MRSA in these populations that limit understanding of its ecology in urban environments. For example, fecal shedding of MRSA, which may increase environmental contamination, has been reported in other species; however, it is unknown whether Norway rats carry the bacteria rectally. Furthermore, while intermittent MRSA shedding has been shown in other species and may dictate when the risk of transmission is highest, duration of carriage has not been examined for Norway rats. Previous work has shown that lethal animal‐control methods may increase the level of pathogens within reservoir populations, possibly by disrupting ecological patterns. However, the impact of rodent‐control on potentially environmentally acquired pathogens like MRSA has not been tested. Using capture‐mark‐recapture methods in an inner‐city neighborhood in Vancouver, Canada, we show that rats intermittently carry MRSA both in the rectum and oropharynx. By assessing the prevalence of MRSA before and after enacting a pest‐control intervention, we report that kill‐trapping had no impact on the prevalence of carriage of this environmentally‐acquired agent.     

Eco‐efficiency as a strategy for optimizing the sustainability of pest management

Agricultural industrialization and the subsequent reliance on pesticides has resulted in numerous unintended consequences, such as impacts upon the environment and by extension human health. Eco‐efficiency is a strategy for sustainably increasing production, while simultaneously decreasing these externalities on ecological systems. Eco‐efficiency is defined as the ratio of production to environmental impacts. It has been widely adopted to improve chemical production, but we investigate the challenges of applying eco‐efficiency to pesticide use. Eco‐efficiency strategies include technological innovation, investment in research and development, improvement of business processes, and accounting for market forces. These components are often part of integrated pest management (IPM) systems that include alternatives to pesticides, but its implementation is often thwarted by commercial realities and technical challenges. We propose the creation and adoption of an eco‐efficiency index for pesticide use so that the broad benefits of eco‐efficient strategies such as IPM can be more readily quantified. We propose an index based upon the ratio of crop yield to a risk quotient (RQ) calculated from pesticide toxicity. Eco‐efficiency is an operational basis for optimizing pest management for sustainability. It naturally favors adoption of IPM and should be considered by regulators, researchers, and practitioners involved in pest management. © 2019 Society of Chemical Industry     

Immunocytochemical localization of the Bacillus sphaericus binary toxin components in Culex quinquefasciatus (Diptera: Culicidae) larvae midgut

This study describes the immunocytochemical localization of the Bacillus sphaericus 2362 binary toxin components, BinA and BinB, in Culex quinquefasciatus larvae that had been intoxicated with this entomopathogen. Ultrathin sections of C. quinquefasciatus larvae midgut embedded in the hydrophilic resin L.R. White were incubated with the antibodies anti-BinA or anti-BinB and then revealed with goat anti-rabbit IgG coupled to gold particles. Immunocytochemical detection demonstrated the presence of specific labeling in ultra-thin sections that had been incubated with the BinA antiserum. Gold particles were detected on the apical areas of cell membranes and inside the epithelial cell cytoplasm, particularly the mitochondria, of cells from the gastric caeca and posterior stomach in larvae exposed during 2 or 24 h to the entomopathogen. A similar labeling pattern was observed in ultrathin sections from both midgut regions when incubated with BinB antiserum.     

Electron microscope observations on the structure of membranes from nuclear polyhedral viruses

Electron micrographs show the presence of the polyhedral, the outer, and the inner membranes occurring in nuclear polyhedral bodies fromMalacosoma neustria, Barathra brassicae andAdoxophyes reticulana. The outer membranes appear to be composed of a central, bimolecular leaflet of lipids bounded on either side by carbohydrates and proteins. The polyhedral and inner membranes, however, seem to consist of protein only. The inner membranes are closely attached to the virus rods, and so are the polyhedral membranes to the polyhedral protein.Samenvatting De polyëdermembranen vanMalacosoma neustria, Barathra brassicae enAdoxophyes reticulana, alsmede de buitenste en binnenste membranen om de deeltjes van deze kernpolyëdervirussen, werden met de elektronenmicroscoop bestudeerd (fig. 1A-I, 2A-C). Om de polyëders te fixeren en ter verkrijging van bepaalde contrasten in ultra-dunne coupes werd van verschillende agentia gebruik gemaakt. Vergelijking van de verkregen foto's (fig. 2D-I, 3A-B) wijst uit, dat het buitenste membraan uit een bimoleculaire laag van lipiden bestaat, die aan beide zijden door koolhydraten en eiwitten zijn begrensd (fig. 4); de polyëdermembranen (fig. 3B) bestaan evenals de binnenste membranen echter uitsluitend uit eiwitten. De virusstaafjes en de polyëders zijn respectievelijk door het binnenste membraan en door het polyëdermembraan nauw omsloten.     

A PCR technique for the detection of Jaagsiekte sheep retrovirus in the blood suitable for the screening of ovine pulmonary adenocarcinoma in field conditions

Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring contagious lung neoplasia caused by jaagsiekte sheep retrovirus (JSRV). Although no specific circulating antibodies against the virus can be detected in infected sheep, JSRV proviral DNA sequences can be found in peripheral blood leukocytes (PBLs) in clinically affected and in a proportion of in contact animals. In this study, existing hemi-nested PCR procedure is compared with a new one-step PCR technique that was developed to minimise potential DNA contamination and reduce sample and reagent handling. Different blood preparations were assessed and the best results were achieved on DNA prepared from buffy coat. The sensitivity of this PCR was lower in JSRV infected sheep without lesions of OPA than in clinically affected sheep, which indicate that this PCR may not be not fully appropriate for screening of individual sheep, but rather to provide results at flock level. This PCR is the only currently available blood test for detection of JSRV infected sheep and may be useful in epidemiological studies and in control programmes of OPA.     

Outcome of desmoplasty and fasciotomy for desmitis involving the origin of the suspensory ligament in horses: 27 cases (1995-2004)

OBJECTIVE: To determine outcome of percutaneous ultrasound-guided desmoplasty with simultaneous fasciotomy for proximal suspensory desmitis (ie, desmitis of the origin of the suspensory ligament) in horses that have not responded to stall rest. DESIGN: Retrospective case series. ANIMALS: 27 horses. PROCEDURES: Medical records of horses with proximal suspensory desmitis treated by means of desmoplasty with fasciotomy were reviewed. Follow-up information was obtained through telephone conversations with owners and trainers of the horses or by examination of horses at the hospital. RESULTS: 23 of the 27 (85%) horses, including 3 of 4 horses with forelimb lesions and 20 of 23 horses with hind limb lesions, were able to return to full work after surgery and rehabilitation. All horses had ultrasonographic evidence of healing of suspensory ligament lesions. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that desmoplasty with fasciotomy is a viable treatment option in horses with proximal suspensory desmitis that have not responded to stall rest.