Further studies on the analysis of DSP toxin profiles in galician mussels

Further studies on mussel samples from Galicia, Spain, have revealed the presence of okadaic acid (OA), dinophysistoxin 2 (DTX2), and the fatty acid acyl esters of both of these toxins as the "DTX3" complex. Measurements were performed with an improved in situ method for the formation of 9-anthryldiazomethane (ADAM) derivatives followed by liquid chromatography with fluorescence detection. Base hydrolysis of DTX3 toxins gave free OA and DTX2, which were determined following ADAM derivatization. Results were confirmed by liquid chromatography/mass spectrometry analyses, and in most of the samples, free DTX2 was the most abundant toxin. However, the OA/DTX2 ratio in the DTX3 conjugated form was different, with OA being the most abundant in all cases. This difference could be due to different rates of metabolism of OA and DTX2 to the acyl esters or due to contamination of the shellfish by the two toxins at different points in time, resulting in less acyl ester formation for one toxin versus the other. The second possibility would be reasonable if two different source organisms were producing the toxins.     

Improved isolation procedure for azaspiracids from shellfish, structural elucidation of azaspiracid-6, and stability studies

Azaspiracids are a group of lipophilic polyether toxins produced by the small dinoflagellate Azadinium spinosum. They may accumulate in shellfish and can result in illnesses when consumed by humans. Research into analytical methods, chemistry, metabolism, and toxicology of azaspiracids has been severely constrained by the scarcity of high-purity azaspiracids. Consequently, since their discovery in 1995, considerable efforts have been made to develop methods for the isolation of azaspiracids in sufficient amounts and purities for toxicological studies, in addition to the preparation of standard reference materials. A seven-step procedure was improved for the isolation of azaspiracids-1-3 (1, 2, and 3) increasing recoveries 2-fold as compared to previous methods and leading to isolation of sufficiently purified azaspiracid-6 (6) for structural determination by NMR spectroscopy. The procedure, which involved a series of partitioning and column chromatography steps, was performed on 500 g of Mytilus edulis hepatopancreas tissue containing ~14 mg of 1. Overall yields of 1 (52%), 2 (43%), 3 (43%), and 6 (38%) were good, and purities were confirmed by NMR spectroscopy. The structure of 6 was determined by one- and two-dimensional NMR spectroscopy and mass spectrometry. The stability of 6 relative to 1 was also assessed in three solvents in a short-term study that demonstrated the greatest stability in aqueous acetonitrile.     

中国沿海两例食用织纹螺中毒事件中织纹螺体内毒素分析

近年来,因食用织纹螺(Nassariusspp.)导致的中毒事件在中国沿海屡有发生。由于中毒患者的症状与麻痹性贝毒中毒症状相似,因此,许多中毒事件被归咎于麻痹性贝毒,认为织纹螺中的毒素与邻近海域的有毒赤潮有关,但也有研究发现螺体内存在河豚毒素。对此,本研究应用亲水性相互作用色谱柱建立了河豚毒素的液相色谱-质谱联用分析方法,对造成2002年和2003年两次中毒事件的织纹螺样品进行分析。结果表明,两批织纹螺样品中均含有高浓度的河豚毒素及其衍生物,包括三脱氧河豚毒素、脱水河豚毒素和单加氧河豚毒素等,而且两个织纹螺样品中的毒素组成非常相似。因此,导致这两起中毒事件的致毒因子是河豚毒素及其衍生物。江苏和福建两地织纹螺中毒素组成的相似性显示两地织纹螺可能具有相同或相近的毒素来源。     

Identification of pinnatoxins and discovery of their fatty acid ester metabolites in mussels ( Mytilus edulis ) from eastern Canada

Pinnatoxins are a group of fast-acting cyclic imine toxins previously identified in shellfish from Asia, the southern Pacific, and northern Europe. In this work pinnatoxins were detected in mussels from locations across the eastern coast of Canada. Pinnatoxin G (6) was the major structural variant present, sometimes at levels >80 μg/kg, whereas much lower levels of pinnatoxin A (1) were detected in some samples. Increased concentrations were observed following base hydrolysis of extracts, leading to the discovery by LC-MS of a range of fatty acid esters of 6. Information on the structures of these acylated derivatives was provided through a series of mass spectrometric experiments, supported by partial synthesis, and it is proposed that the compounds are 28-O-acyl esters of 6. Although acyl esters of a range of other phycotoxins are known to form as metabolites in shellfish, this is the first report of their existence for this particular toxin class. The occurrence of pinnatoxins in North American shellfish further highlights the international distribution of these toxins.     

Post‐exposure rabies prophylaxis for mass bat exposures: Case series and systematic review

To assess and describe the use of rabies post‐exposure prophylaxis (PEP) related to mass bat exposures (MBEs) in the literature and in the state of Rhode Island. Data on MBE events occurring between 2010 and 2016 from the Rhode Island Department of Health (RIDOH) were reviewed to determine PEP usage. For comparison, a systematic review was also performed by searching PubMed, EMBASE, Cochrane Library and Web of Science using the keywords ‘rabies,’ ‘bat’ and ‘exposure.’ There were three MBE events in Rhode Island between 2010 and 2016 in which groups of individuals were asleep during the potential exposure. As a result, RIDOH provided PEP to those involved in the event. In a systematic review of 12 published reports, a variety of settings were involved in MBEs: camping sites, a domestic flight, a neonatal intensive care unit and dormitories. The number of potential rabies exposures ranged from five to 1,429 people. PEP recommendations for assessed persons ranged from 0% to 100% across events (median 21%). The variation in PEP recommendations following a MBE may be dependent on medical need, past precedent or preference. Federal guidance for MBE is needed to add clarity and to minimize the variability in PEP recommendations for such events in the future.     

Using a modified ferrous oxidation-xylenol orange (FOX) assay for detection of lipid hydroperoxides in plant tissue.

The ferrous oxidation-xylenol orange (FOX) assay was adapted for quantifying lipid hydroperoxides (LOOHs) in plant extracts. Excised pieces of several fruit and vegetable species were exposed to 83 kJ m(-2) day(-1) of biologically effective ultraviolet B irradiance (UV-B(BE)) for 10-12 days to induce cellular oxidation. The LOOH and thiobarbituric acid reactive substance (TBARS) concentrations of these plant tissues were assessed with the FOX and iodometric assays for the former and a modified TBARS assay for the latter. There was generally good agreement between the FOX and iodometric methods both prior to and following the UV exposure. However, the iodometric assay appeared to have some difficulty in consistently quantifying lower LOOH levels (<11 microM), whereas the FOX assay measured LOOH concentrations as low as 5 microM. All tissues exhibited UV-induced increases in TBARS, indicating a marked degree of cellular oxidation in the exposed tissue segments. Compared with the iodometric assay, the FOX method consistently generated less variable LOOH values. The presence of authentic linoleic acid-OOHs in spiked avocado and spinach samples (11 microM) was identified with liquid chromatography-mass spectrometry techniques, which validated corresponding FOX assay results. The FOX method is inexpensive, is not sensitive to ambient O2 or light levels, and can rapidly generate LOOH measurements. The physiological value of the FOX assay resides in its ability to measure initial rather than more advanced fatty acid oxidation; hence, early membrane-associated stress events in plant tissue can be detected.     

Zinc from oyster tissue as causative factor in mouse deaths in official bioassay for paralytic shellfish poison

Toxicity (extreme weakness, body temperature drop, cyanosis, some slow deaths) in test mice, upon intraperitoneal injection of standard-method paralytic shellfish poison (PSP) extracts of some PSP-free oysters, is consistent with the relatively high levels of zinc in these extracts. As a rough guideline, the threshold for a toxic response corresponds to a drained tissue zinc level of over 900 micrograms/g. The identification of zinc as the substance responsible has been supported by inducing toxicity in control extracts by spiking with nontoxic levels of zinc, and by eliminating toxicity from toxic extracts by chemical removal (precipitation, ion exchange) of metals.     

Influence of Land Use and Nutrient Flux on Metabolic Activity of E. coli O157 in River Water

Infections caused by waterborne Escherichia coli O157 continue to pose a public health risk. An increase in faecal coliform loading of watercourses due to expanding populations, intensification of agriculture and climate change are predicted to amplify these risks. Understanding the effect of land use on the ecology of E. coli O157 in environmental waters is therefore important for implementing effective mitigation measures. In order to test the hypothesis that activity of waterborne E. coli O157 is affected by both land use type and the respective autochthonous microbial communities, we inoculated replicate microcosms of water collected from areas of contrasting land uses within a catchment with a chromosomally lux-marked E. coli O157. Pathogen metabolic activity and its ability to reactivate following addition of nutrients were quantified over time in both filter-sterilised and non-sterile microcosms. Metabolic activity differed significantly according to the land use type, the degree of competition from background microbes and the availability of nutrients. These results indicate that land use types associated with particular areas of a watercourse should be considered a central factor in models that aim to predict pathogen risk in environmental waters.     

Seroprevalence and risk factors associated with Escherichia coli O157 in a farming population

Evidence suggests that frequent and direct exposure to domestic animals has made farmers less susceptible to symptomatic Escherichia coli O157 infection than other members of the community. We have quantified the seroprevalence of antibodies to E. coli O157 lipopolysaccharide (LPS) from a representative cohort of farm workers in three geographically distinct regions of the United Kingdom during two sampling rounds over a 2-year period. All participants completed a questionnaire to determine the range and extent of recent animal contact alongside other potential occupational and environmental exposure routes. A total of 31/946 (3.3%) serum samples contained antibodies to E. coli O157 LPS (from both rounds combined). On the second sampling round, a significant difference in seropositivity was apparent between the three regions, with enhanced seroprevalence linked to recent contact with beef cattle, having a private water supply and contact with a child under 5 years old. Only five seropositive people reported symptoms of a gastrointestinal tract infection, although these symptoms were mild. These results further support the premise of acquired immunity to E. coli O157 associated with prolonged antigenic exposures within the farming environment.     

Haustoria of microcyclic rust fungi Uromyces ficariae and Puccinia tumida and other gall-forming species, U. dactylidis (macrocyclic) and P. smyrnii (demicyclic)

Cytological studies involving light and scanning electron microscopy examined the morphology of intracellular hyphae of various gall-forming species of rust fungi. Uromyces ficariae (microcyclic) and U. dactylidis (macrocyclic, heteroecious) both produced vermiform and largely indeterminate intracellular hyphae typical of M-haustoria, associated with telial and aecial galls, respectively, on their common host Ranunculus ficaria . Similar M-haustoria were observed in telial galls of Puccinia tumida (microcyclic) on Conopodium majus , in host tissue affected by nine other microcyclic species and in aecial galls of P. smyrnii (demicyclic, autoecious) on Smyrnium olusatrum . Telia of P. smyrnii , however, infecting photosynthetically active mesophyll without gall formation, produced typical D-haustoria with determinate growth, a narrow neck region and a bulbous clavate body. Gall formation was associated with hyphal infection of vascular tissue. The relationship between M-haustoria, vascular infection and gall formation is discussed in relation to the evolution of microcyclic rusts and Tranzschel's law.