Isolation and Characterization of Microcystin-Degrading Bacteria from Lake Erie

Heterotrophic bacteria are suggested as the major agents that degrade microcystins (MCs), a major cyanotoxins, in natural environments. However, little is known of the taxonomic and functional diversity of MC-degrading bacteria in Lake Erie of the Laurentian Great Lakes, the largest freshwater system on earth. This study obtained six bacterial pure isolates from Lake Erie with an ability to use MCs as the sole carbon and energy sources. MC degradation rates of the isolates were impacted by temperature and pH. The key gene for MC degradation (mlrA) were failed to be PCR amplified from for all 6 MC degraders, indicating they may possess a novel MC degradation pathway. In addition for potentials used in MC bioremediation, two isolates maybe can offer extra benefits as biofertilizers.     

Allelopatic effects of cyanobacteria extracts containing microcystins on Medicago sativa-Rhizobia symbiosis

The eutrophication of water leads to massive blooms of cyanobacteria potentially producers of highly toxic substances: cyanotoxins, especially microcystins (MC). The contamination of water used for irrigation by these toxins, can cause several adverse effects on plants and microorganisms. In this work, we report the phytotoxic effects of microcystins on the development of symbiosis between the leguminous plant Medicago sativa (Alfalfa) and rhizobia strains. The exposure of rhizobial strains to three different concentrations 0.01, 0.05 and 0.1 μg MC ml⁻¹ led to decrease on the bacteria growth. The strains of rhizobia Rh L1, Rh L2, Rh L3 and Rh L4 reduced their growth to, respectively, 20.85%, 20.80%, 33.19% and 25.65%. The chronic exposure of alfalfa seeds and seedlings to different MC concentrations affects the whole stages of plant development. The germination process has also been disrupted with an inhibition, which reaches 68.34% for a 22.24 μg MC ml⁻¹. Further, seedlings growth and photosynthetic process were also disrupted. The toxins reduced significantly the roots length and nodule formation and leads to an oxidative stress. Thus, the MCs contained in lake water and used for irrigation affect the development of symbiosis between M. sativa and Rhizobia.     

微囊藻毒素毒理学的研究进展

水体富营养化现象日益严重及藻类水华的频繁暴发已成为全球广泛关注的环境问题.而微囊藻毒素(Microcystin,MCs)是在蓝藻水华污染中出现频率最高、造成危害最严重的一种环七肽肝毒素.目前,蓝藻水华的研究主要集中在MCs对水产动物及哺乳动物造成的伤害.大量研究表明,作为肝毒素的MCs具有稳定性,不但能够在生物链中积累...     

鱼腥藻毒素与微囊藻毒素的免疫毒性研究

目的蓝藻水华暴发日益频繁,蓝细菌滋生、产生大量有毒代谢产物——藻毒素。最典型的有鱼腥藻毒素-A(Anatoxin-a,AnTX-a)和微囊藻毒素(Microcystins,MC)。AnTX-a是一种快速致死性神经毒素,MC是普遍存在的细胞和肝毒素,被这些毒素污染的水体和食物会给人畜健康带来严重危害,也可能对人群公共健康造成威胁。方法通过查阅国内外研究进展,概述了AnTX-a和MC对机体免疫系统的毒性作用。结果近年来大量研究表明,AnTX-a和MC对机体的危害不仅仅限于神经系统和实质器官,还能对人类、动物以及水生生物中免疫细胞和免疫分子产生毒性效应。结论此综述从免疫学角度,为深入探讨AnTX-a和MC对机体的联合免疫毒性作用和机理提供新的视野和思路。     

Do main partner conflict, power dynamics, and control over use of male condoms predict subsequent use of the female condom?

This study assessed hypotheses that measures of power and control over male condom (MC) use would predict use of the female condom (FC) among women with main partners from two public STD clinics (n = 616). The women (mean age 24 years, 87% African American) were enrolled in an intervention study to promote barrier contraceptive use and were interviewed at baseline and at 6 monthly follow-up visits. Seven baseline predictor variables were assessed: her having requested MC use, his having objected, her having wanted a MC used but not asking, percentage of MC use, perceived control over MC use, anticipated consequences of refusing unprotected sex, and physical violence. In the first Poisson regression analysis, none of the hypothesized predictors was significantly associated with FC use during follow up. In the second regression analysis, which assessed the influence of the hypothesized set of predictors on follow-up FC use in situations when MCs were not used, we found two effects. Either no or inconsistent MC use before study entry was associated with less subsequent FC use; women who reported, at study entry, having more control over MC use were more likely to use FCs during follow up. We found no evidence of adoption of the FC by women in relationships marked by history of conflict over the MC, circumstances in which alternatives are most needed. On the contrary, we found that women with a history of control and consistent use of MCs were the most likely users of FCs when MCs were not used.     

Self discipline and obesity in Bangkok school children

Background

In 2004, the Ministry of Health issued the policy of decentralising microscopy services (MCs) to one third of all township hospitals in China. The study was conducted in Gansu Province, a poor western one in China. Ganzhou was one county in Gansu Province. Ganzhou County was identified as a unique case of further decentralisation of tuberculosis (TB) treatment services in township hospitals. The study evaluated the impact of the MC policy on providers and patients in Gansu Province. The second objective was to assess the unique case of Ganzhou County compared with other counties in the province.

Methods

Both quantitative and qualitative methods were used. All 523 MCs in the province completed an institutional survey regarding their performance. Four counties were selected for in-depth investigation, where 169 TB suspects were randomly selected from the MC and county TB dispensary registers for questionnaire surveys. Informant interviews were conducted with 38 health staff at the township and county levels in the four counties.

Results

Gansu established MCs in 39% of its township hospitals. From January 2006 to June 2007, 8% of MCs identified more than 10 TB sputum smear positive patients while 54% did not find any. MCs identified 1546 TB sputum smear positive patients, accounting for 9% of the total in the province. The throughputs of MCs in Ganzhou County were eight times of those in other counties. Interviews identified several barriers to implement the MC policy, such as inadequate health financing, low laboratory capacity, lack of human resources, poor treatment and management capacities, and lack of supervisions from county TB dispensaries.

Conclusion

Microscopy centre throughputs were generally low in Gansu Province, and the contribution of MCs to TB case detection was insignificant taking account the number of MCs established. As a unique case of full decentralisation of TB service, Ganzhou County presented better results. However, standards and quality of TB care needed to be improved. The MC policy needs to be reviewed in light of evidence from this study.     

Occupational and environmental hazard assessments for the isolation, purification and toxicity testing of cyanobacterial toxins

Cyanobacteria can produce groups of structurally and functionally unrelated but highly potent toxins. Cyanotoxins are used in multiple research endeavours, either for direct investigation of their toxicologic properties, or as functional analogues for various biochemical and physiological processes. This paper presents occupational safety guidelines and recommendations for personnel working in field, laboratory or industrial settings to produce and use purified cyanotoxins and toxic cyanobacteria, from bulk harvesting of bloom material, mass culture of laboratory isolates, through routine extraction, isolation and purification. Oral, inhalational, dermal and parenteral routes are all potential occupational exposure pathways during the various stages of cyanotoxin production and application. Investigation of toxicologic or pharmacologic properties using in vivo models may present specific risks if radiolabelled cyanotoxins are employed, and the potential for occupational exposure via the dermal route is heightened with the use of organic solvents as vehicles. Inter- and intra-national transport of living cyanobacteria for research purposes risks establishing feral microalgal populations, so disinfection of culture equipment and destruction of cells by autoclaving, incineration and/or chlorination is recommended in order to prevent viable cyanobacteria from escaping research or production facilities.     

The effect of cyanobacterial blooms in the Siemianówka Dam Reservoir on the phytoplankton structure in the Narew River

The effect of cyanobacterial blooms on the phytoplankton structure in the lowland Narew River (north-eastern Poland) was examined. The studies were carried out at stations situated at different distances from the eutrophic Siemianówka Dam Reservoir. In 2008, the investigated lowland reservoir and the outflowing river were characterized by the dominance of toxic cyanobacteria (from July to October). At a station situated 130 km below the dam, species composition in the river was very similar to that in the shallow reservoir. Planktothrix agardhii was the main and permanent dominant, both in limnoplankton and potamoplankton. The current study indicates that the eutrophic Siemianówka Dam Reservoir is the main and rich source of phytoplankton for the outflowing Narew River. Cyanobacteria were dominant in the river phytoplankton at all sampling stations, but their share in phytoplankton biomass gradually decreased with the distance from the dam. Chemical analysis revealed the presence of microcystins (MCs) in water samples collected from June to October. The toxins, mainly demethylated MC analogues, were detected at a long distance down the river, even 100 km from the dam. Maximum concentration of MCs (14.3 μg l^?1) was measured on 13 October, 9.1 km below the dam.     

淀山湖夏秋季微囊藻毒素-LR和类毒素-A分布状况及其影响因素

为了解淀山湖夏秋季藻类和藻毒素分布状况及其影响因素 ,在淀山湖选了 6个点 ,分别测定了总氮、总磷、铁、叶绿素 -A和COD的含量 ,同时记录了水温、pH值、照度、浊度 ,另外还进行了藻细胞计数和藻种的鉴定以及微囊藻毒素 -LR和类毒素 -A含量的测定。结果表明 ,两种藻毒素均在 7月份污染最严重 ,微囊藻毒素 -LR在网箱分布最多 ,而类毒素 -A在急水港分布最多。广义估计方程回归分析表明 ,藻细胞总数与总氮、总磷和叶绿素 -A有显著的相关性 (P <0 0 5) ,藻细胞总数和水温对微囊藻毒素 -LR的分布有显著的影响 (P <0 0 1 ) ,水体浊度可显著影响类毒素 -A的分布 (P <0 0 1 )。微囊藻毒素 LR与类毒素 -A有轻度的相关关系 (P <0 0 5)。总之 ,淀山湖存在较严重的微囊藻毒素 -LR污染和较轻度的类毒素 -A污染 ,影响两种毒素分布的因素并不完全一致     

The effects of blue algae on health

Cyanobacteria (blue algae) regularly cause recreational waters to become murky and smelly. Skin irritation and mild gastrointestinal disorders have regularly been reported following recreational activities in water suspected of being contaminated with cyanobacteria. The exact cause of these effects on health is not clear. Severe effects are not to be expected from recreational exposure to water contaminated with cyanobacteria. Cyanobacteria can produce hepatotoxins, neurotoxins, cytotoxins and irritants. In Brazil lethal intoxications have occurred due to the occurrence of toxins in drinking water and in dialysis fluid. The Dutch policy is based on the Commissie Integraal Waterbeheer (Commission Integral Water Management) guidelines for recreational waters. It is not clear to what extent the other cyanotoxins occur in the Netherlands. However, several genera ofcyanobacteria capable of producing these other cyanotoxins have been found in the Netherlands. For a good risk assessment in the Netherlands, more information is needed on the effects on health of cyanobacteria. There is also a need for more data on the prevalence of different cyanobacteria and toxins in Dutch recreational waters.     

Cyanobacteria and cyanotoxins in Polish freshwater bodies

In this work, the authors examined the presence of cyanobacteria and cyanotoxins in 21 samples collected from fresh water bodies located in 5 provinces in Poland: Lublin (2), Podlasie (1), Pomerania (6), Warmia-Masuria (1) and Wielkopolska (11). In addition, to determine the general pattern of geographical distribution, frequency of cyanobacteria occurrence, and cyanotoxins production, the published data from 238 fresh water bodies in Poland were reviewed. On the basis of these collected results, we concluded that Planktothrix, Aphanizomenon, Microcystis and Dolichospermum were dominant. The general pattern in geographical distribution of the identified cyanobacterial genera was typical of other eutrophic waters in Europe. The production of cyanotoxins was revealed in 18 (86%) of the 21 samples analyzed in the present work and in 74 (75%) of the 98 total water bodies for which the presence of toxins had been examined. Among the 24 detected microcystin variants, [Asp3]MC-RR was most common. These results can be verified when more data from the less explored water bodies in the southern and eastern parts of Poland are available.     

Mast cells and γδ T cells are largely dispensable for adaptive immune responses after laser-mediated epicutaneous immunization

BackgroundThe skin resembles an attractive target for vaccination due to its accessibility and abundance of resident immune cells. Cells like γδ T cells and mast cells (MCs) are part of the first line of defence against exogenous threats. Despite being important mediators for eliciting TH2 immune responses after epithelial stress, γδ T cell and MC functions still remain to be completely understood. Here, we aimed to characterize their roles in shaping adaptive immune responses after laser-mediated epicutaneous immunization (EPI).Methodsγδ T cell knock out, MC-depleted, and wildtype control mice were immunized with mannan-conjugated grass pollen allergen Phl p 5 (P5-MN) by laser-mediated EPI. After 2–3 immunizations, cytokine expression, T helper polarization, and antigen-specific IgG1/IgE levels were analysed. Furthermore, the local cytokine/chemokine milieu after laser microporation was determined.ResultsThe majority of inflammatory chemokines and cytokines induced by laser treatment were not affected by the presence of γδ T cells or MCs. However, RANTES was elevated in γδ T cell knock out mice and GROα, TSLP, and IL-33 were significantly decreased after MC depletion. The absence of γδ T cells or depletion of MCs had no substantial effect on adaptive immune responses after laser-mediated EPI, except for slightly reduced IgG1 and effector T cell levels in MC-depleted mice.Conclusionsγδ T cells did not play a pivotal role in shaping the humoral and cellular adaptive immune response after laser-mediated EPI. MC depletion decreased the numbers of effector T cells, indicating a potential role of MCs in the activation and maturation of T cells after EPI.     

Viral antigen production in cell cultures on microcarriers Bovine parainfluenza 3 virus and MDBK cells

Viral antigens can be obtained from infected mammalian cells cultivated on microcarriers. We have worked out parameters for the production of bovine parainfluenza 3 (PI-3) virus by Mandin-Darby Bovine Kidney (MDBK) cells cultivated on Cytodex 1 microcarriers (MCs) in spinners flasks and bioreactor using fetal bovine serum (FBS) supplemented Eagle minimal essential medium (Eagle-MEM). Medium renewal during the cell culture was shown to be crucial for optimal MCs loading (>90% MCs with confluent cell monolayers) and cell growth (2.5 x 10(6)cells/mL and a micro(x) (h(-1)) 0.05). Since cell cultures performed with lower amount of MCs (1g/L), showed good performances in terms of cell loading, we designed batch experiments with a lower concentration of MCs in view of optimizing the cell growth and virus production. Studies of cell growth with lower concentrations of MCs (0.85 g/L) showed that an increase in the initial cell seeding (from 7 to 40 cells/MC) led to a different kinetic of initial cell growth but to comparable final cell concentrations ((8-10)x10(5)cells/mL at 120 h) and cell loading (210-270 cells/MC). Upon infection with PI-3 virus, cultures showed a decrease in cell growth and MC loading directly related to the multiplicity of infection (moi) used for virus infection. Infected cultures showed also a higher consumption of glucose and production of lactate. The PI-3 virus and PI-3 antigen production among the cultures was not significantly different and attained values ranging from, respectively, 7-9 log(10) TCID(50)/mL and 1.5-2.2 OD. The kinetics of PI-3 virus production showed a sharp increase during the first 24h and those of PI-3 antigen increased after 24h. The differential kinetics of PI-3 virus and PI-3 antigen can be explained by the virus sensitivity to temperature. In view of establishing a protocol of virus production and based on the previous experiments, MDBK cell cultures performed under medium perfusion in a bioreactor of 1.2L were infected and the PI-3 virus production in 12L attained 12 log(10) TCID(50). Other than establishing a protocol for PI-3 production in MDBK cell cultures on Cytodex 1, the experiments are proposed as a basis for approaching the development of a virus production protocol in mammalian cells cultivated on microcarriers in bioreactors.     

藻毒素对实验性大鼠肝癌的促进作用

目的 研究藻毒素在实验性大鼠肝癌诱导过程中的促进作用。方法 以二乙基亚硝胺(DEN)为启动剂，藻毒素为促进剂，构建大鼠肿瘤促进模型(Solt-Farber模型)，观察肝脏病理形态改变，分别运用免疫组化法和RT-PCR、原位杂交法检测肝细胞中GSTPi蛋白和GSTPi mRNA表达情况。结果 实验组动物肝脏产生结节性增生灶，藻毒素单独作用不能诱导GSTPi mRNA及蛋白表达，但能够促进DEN诱导产生的GSTPi mRNA及蛋白表达，使其表达增加，并具有一定的剂量-反应关系。结论 藻毒素对实验性大鼠肝癌的发生具有促进作用，提示具有促癌活性。     

Necessity of Screening Water Chestnuts for Microcystins After Cyanobacterial Blooms Break Out

Water chestnut is one of the most popular vegetables in Asian countries that grows in shallow water. Eighteen water chestnut samples were collected from Lake Tai and six samples were bought at markets in Wuxi, China, in October 2007. Extraction solution of water chestnut was cleaned up with a solid phase extraction column and immunoaffinity chromatography cartridges, then the microcystin (MC) level was detected by indirect competitive enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-mass spectrometry (LC-MS). The results of ELISA showed that there were six samples collected from Lake Tai which contained MCs; the highest level of total MCs was 7.02 ng/g. The results of LC-MS confirmed that MC-LR and MC-RR were present in five samples. The highest level of MC-LR was 1.02 ng/g and that of MC-RR was 4.44 ng/g. Heavy cyanobacterial blooms had occurred, and MCs were detected in water at the points in Lake Tai where MCs occurred in water chestnuts collected in 2007. MCs were not detected in the six samples bought at Wuxi markets. The results suggest that MCs can accumulate in water chestnuts, which is a potential hazard for human health.     

Influence of calcium and silicon supplementation into Pleurotus ostreatus substrates on quality of fresh and canned mushrooms

Supplements of gypsum (calcium source), pumice (silicon source) and pumice sulfate (silicon and calcium source) into substrates for oyster mushrooms (Pleurotus ostreatus) were searched for their effects on production as well as qualities of fresh and canned mushrooms. The addition of pumice up to 30% had no effect on total yield, size distribution and cap diameters. The supplementation of gypsum at 10% decreased the total yield; and although gypsum at 5% did not affect total yield, the treatment increased the proportion of large-sized caps. High content (>10%) of pumice sulfate resulted in the lower yield. Calcium and silicon contents in the fruit bodies were not influenced by supplementations. The centrifugal drip loss values and solid content of fresh mushrooms, and the percentage of weight gained and firmness of canned mushrooms, cultivated in substrates supplemented with gypsum, pumice and pumice sulfate were significantly (p≤0.05) higher than those of the control. Scanning electron micrographs revealed the more compacted hyphae of mushroom stalks supplemented with silicon and/or calcium after heat treatment, compared to the control. Supplementation of P. ostreatus substrates with 20% pumice was the most practical treatment because it showed no effect on yield and the most cost-effective.     

Blood and mast cell histamine levels in magnesium-deficient rats

The number and morphology of mast cells (MCs) in the duodenal submucosa and the histamine content of blood and isolated peritoneal mast cells were estimated sequentially in male weanling Sprague-Dawley rats magnesium (Mg)-depleted for up to 48 days. Blood histamine levels increased 4--5-fold by 14 days of Mg depletion and subsequently declined to levels similar to those in pair-fed Mg-adequate controls. In the same period, the submucosal MCs continuously increased in number, beginning to plateau at 6--7 times the control number between days 36--48 of Mg deficiency. Massive MC degranulation was seen in the early stages of Mg-depletion but subsided after 16--20 days. Subsequently, the submucosal MCs appeared small and rounded. They showed reduced staining with toluidine blue indicating a deficiency in the mucopolysaccharide component of the storage granules. Histamine content of purified peritoneal MCs from Mg-deficient rats was reduced by 10 days of Mg depletion and remained at or below one-third the control level throughout the depletion period. The data suggest that the MCs remaining or developed during chronic Mg depletion are deficient in their capacity to store and secrete histamine.     

Effects of microcystins on fish

Microcystins (MCs) are hepatotoxic heptapeptides released into water during or on senescence of cyanobacterial blooms. This review details the different effects of the MCs on fish and discusses their potential consequences in aquatic food webs. In early life stages, exposure to MCs causes, in a dose-dependent manner, perturbations to embryonic hatching, decrease in survival and growth rate, as well as histopathological effects (enlarged and opaque yolk sac, small head, curved body and tail, hepatobiliary abnormalities, ultrastructural alterations in hepatocytes, heart rate perturbations). In adults and juveniles, field and experimental studies demonstrated that after ingestion MCs accumulate mainly in liver but can also be found in muscle and viscera. Microcystin exposure has been shown to affect growth rate and osmoregulation, increase liver enzyme activities in the serum and heart rate, modify behavior, and exert histopathological effects in the liver, intestine, kidneys, heart, spleen, or gills, but the degree to which these effects were seen depends on the exposure route. The detoxication pathway of MCs in fish begins with a conjugation reaction to glutathione catalyzed by glutathione S-transferases, and this is comparable to the reaction demonstrated in other organisms, from plants to mammals. It appears that MC concentrations found in nature can potently affect several trophic levels in the aquatic ecosystems, in particular by inducing failure of sensitive stages (e.g., fish fry) to develop and accumulating in the food chains. The need of further quantitative studies on the sublethal effects, accumulation, and fate of MCs in aquatic food chains still remains.     

Detection of potentially producing cylindrospermopsin and microcystin strains in mixed populations of cyanobacteria by simultaneous amplification of cylindrospermopsin and microcystin gene regions

Cyanobacterial blooms are frequently formed by heterogeneous populations of toxin-producing and non-producing strains. Microcystins (MC) and cylindrospermopsin (CYN) are the most representative cyanobacterial toxins. We have developed a multiplex PCR assay that allows simultaneous detection of MC(+) and/or CYN(+) strains in mixed populations of cyanobacteria. Various primer sets were designed using mcy and aoa gene sequences related with MC and CYN synthesis respectively, to amplify at the same time aoa and mcy sequences. Purified DNA, cultured cell mixtures and field samples with MC and CYN producing strains were used as DNA template. The results show: (i) the expected amplicons were only observed with toxic strains; (ii) cells were suitable as a source of purified DNA for the multiplex PCR; (iii) the assay could detect simultaneously 3 aoa and 3 mcy gene regions with mixed CYN(+) and MC(+) cyanobacteria cells. The method could be applied to environmental samples, allowing in a rapid, economical and easy way to detect simultaneously the presence of CYN(+) and MC(+) cyanobacteria in sestonic fractions of water samples.     

Effects of cyanobacterial toxins and cyanobacterial cell-free crude extract on germination of alfalfa (Medicago sativa) and induction of oxidative stress

Cyanobacterial toxins have adverse effects on both terrestrial and aquatic plants. Microcystins are cyclic heptapeptides and an important group of cyanotoxins. When lake water contaminated with cyanobacterial blooms is used for spray irrigation, these toxins can come in contact with agricultural plants. During the exposure to these toxins, reactive oxygen species can form. These reactive oxygen species have a strong reactivity and are able to interact with other cellular compounds (lipids, protein, and DNA). Plants have antioxidative systems that will limit the negative effects caused by reactive oxygen species. These systems consist of enzymes, such as superoxide dismutase, catalase, and ascorbate peroxidase, and nonenzymatic substances, such as reduced glutathione or vitamins. The aim of the present study was to investigate the effects of cyanobacterial toxins (microcystins and anatoxin-a) and cyanobacterial cell-free crude extract on alfalfa (Medicago sativa) seedlings. Inhibition of germination and root growth was observed with toxin concentrations of 5.0 microg/L. Also, oxidative damage, such as lipid peroxidation, was detected after the exposure of alfalfa seedlings to the toxin. Reactive oxygen detoxifying enzymes were elevated, showing a marked response in alfalfa to oxidative stress caused by the exposure to cyanobacterial metabolites that might influence the growth and development of these plants negatively.