Biodiversity of Clostridium botulinum type E associated with a large outbreak of botulism in wildlife from Lake Erie and Lake Ontario

The genetic relatedness of Clostridium botulinum type E isolates associated with an outbreak of wildlife botulism was studied using random amplification of polymorphic DNA (RAPD). Specimens were collected from November 2000 to December 2008 during a large outbreak of botulism affecting birds and fish living in and around Lake Erie and Lake Ontario. In our present study, a total of 355 wildlife samples were tested for the presence of botulinum toxin and/or organisms. Type E botulinum toxin was detected in 110 samples from birds, 12 samples from fish, and 2 samples from mammals. Sediment samples from Lake Erie were also examined for the presence of C. botulinum. Fifteen of 17 sediment samples were positive for the presence of C. botulinum type E. Eighty-one C. botulinum isolates were obtained from plants, animals, and sediments; of these isolates, 44 C. botulinum isolates produced type E toxin, as determined by mouse bioassay, while the remaining 37 isolates were not toxic for mice. All toxin-producing isolates were typed by RAPD; that analysis showed 12 different RAPD types and multiple subtypes. Our study thus demonstrates that multiple genetically distinct strains of C. botulinum were involved in the present outbreak of wildlife botulism. We found that C. botulinum type E is present in the sediments of Lake Erie and that a large range of bird and fish species is affected.     

An outbreak of type C botulism in herring gulls (Larus argentatus) in southeastern Sweden

From 2000 to 2004, over 10,000 seabirds, primarily Herring Gulls (Larus argentatus), died from an undetermined cause in the Blekinge archipelago in southeastern Sweden. In June 2004, 24 affected Herring Gulls were examined clinically, killed humanely, and 23 were examined by necropsy. Seven and 10 unaffected Herring Gulls collected from a local landfill site and from Iceland, respectively, served as controls. All affected birds showed similar neurologic signs, ranging from mild incoordination and weakness to severe flaccid paralysis of legs and wings, but generally were alert and responsive. All affected gulls were in normal nutritional condition, but were dehydrated and had empty stomachs. No gross or microscopic lesions, and no bacterial or viral pathogens were identified. Type C botulinum toxin was detected in the sera of 11 of 16 (69%) affected gulls by mouse inoculation. Type C botulism was the proximate cause of disease in 2004. Sera from 31% of birds tested from outbreaks in 2000 to 2003 also had detectable type C botulinum toxin by mouse inoculation. No large-scale botulism outbreak has been documented previously in this area. The source of toxin, initiating conditions, and thus, the ultimate cause of this outbreak are not known. This epidemic might signal environmental change in the Baltic Sea.     

Occurrence ofClostridium botulinum in the soil of the vicinity of Rome

After the isolation of two strains ofClostridium butyricum that produced type E botulinal toxin from the feces of two babies in Rome affected by infant botulism (P. Aureli, L. Fenicia, M. Gianfranceschi, L. McCroskey, and C. Hatheway, J Infect Dis 154:207–211, 1986), extensive research was carried out to detect botulinal toxin-producing clostridia in soil samples from the vicinity of Rome. A total of 520 specimens from cultivated and pasture lands, obtained from 52 collection stations, were examined.Enrichment cultures were tested for botulinal toxin, and the isolated neurotoxigenic micro-organisms were characterized both enzymatically and gas chromatographically.Clostridium botulinum type A and B proteolytic, the former being the most common, were found in 7 (1.3%) out of 520 samples from 5 (9.6%) of the 52 locations in close association with pasture lands. Possible relationships between the presence of botulinal organisms and the properties of the soil in which they were found have also been investigated.     

Persistence of specific antigenic protein in the serum of chickens given intravenously botulinum toxin type B, C, D, E or F

A sublethal dose of Clostridium botulinum progenitor toxin of each of types B, C, D, E, and F was injected once intravenously into chickens. Blood samples were withdrawn periodically from the chickens to determine the toxin remaining in the serum by the mouse injection test and by enzyme-linked immunosorbent assay (ELISA) for both toxic and nontoxic components composing the progenitor toxin. Both components were detected by ELISA for at least a few days after the serum had became innocuous to mice, indicating a higher stability of the antigenicities of both components than the lethal toxicity in the chicken serum. For the diagnosis of botulism, it seems justified to recommend detection of the antigen (toxic component or nontoxic component or both) by ELISA even if no toxin is detected by the mouse test. Such immunological tests would no doubt contribute to an increase in the rate of diagnosis of human and animal botulism cases, particularly when blood sampling is delayed.     

Dolichopodid Biodiversity and Site Quality Assessment of Reed Marshes And Grasslands in Belgium (Diptera: Dolichopodidae)

The conservation value of grassland and reed marsh habitats in Belgium was determined on the basis of dolichopodid communities (Diptera: Dolichopodidae). Four grassland and one reed marsh sites within the 'Bourgoyen-Ossemeersen' Nature Reserve (B.O.) (Ghent) were sampled with white water traps from March 1993 until March 1994. Subsequently, these soil faunas were compared with those of reed marsh habitats in 'Het Meetjeslandse Krekengebied' (M.K.) sampled in 1990. Comparisons of species communities were carried out by means of multivariate analysis techniques. The DCA and CCA on the B.O. sites produced a clear separation between the grassland and the reed marsh communities. Light intensity and soil humidity were among the most important factors determining species distributions. The comparison between the B.O. and M.K. communities revealed that sampling sites were clustered according to their geographical location rather than to habitat type. Overall species richness and diversity was not significantly different between grasslands and reed marshes. However, conservation quality estimates of the sites using the Site Conservation Quality Index (SCQI) indicated that reed marshes comprise a considerably larger number of rare and Red Data Book species. Moreover, besides typical reed marsh-inhabiting species, reed marshes also house species with other ecological affinities sometimes in rather high abundance. As a result, reed marshes are considered highly valuable for the survival of several wetland dolichopodid species.     

Botulism: Laboratory Methods and Epidemiology

Although food botulism (FB) in Argentina was described by 1911, the first documented outbreak was recorded in 1922. In 1957, an outbreak of type A FB caused by red bell peppers was the first laboratory confirmation of botulism in Argentina. From 1922 to 1997, 70 FB outbreaks affecting 242 persons with 111 deaths (case fatality rate, 46%) were reported in Argentina. Infant botulism (IB) was recognized in 1976 and has been mostly diagnosed in the U.S.A. More than 146 IB cases have been reported in Argentina since 1982. Additional cases may go undiagnosed due to physician inexperience and limited access to diagnostic services. A single laboratory-confirmed case of wound botulism (WB) occurred in Argentina in 1995. The botulinal neurotoxins (BoNTs) identified in Argentina have been types A, B, E, F and Af in FB, and exclusively type A in IB and WB.For the laboratory diagnosis of botulism, serum, gastrointestinal sample, food, and wounds should be tested for BoNT. Gastrointestinal, wound and food sample must also be cultured for toxigenic organisms. When higher volumes of serum were tested, BoNT was found in 61% of IB patients in Mendoza compared with 13% in a previous series from the U.S.A.Reliable typing can only be achieved when the BoNT belongs to a known serotype and the toxin titer is above 4000 LD₅₀/mL. When these criteria are not met, as in most clinical samples, bacterial isolation, purification and adequate toxin production in culture are required. Neutralization testing must be performed at not less than three 10-fold doses of toxin because of (1) the existence of subtypes, where a second, minor serotype is present, (2) the sharing of epitopes between certain serotypes, and (3) the occurrence of serological variants. Three basic properties of working antitoxins, specificity, protency and avidity, must be known for BoNT typing. The efficiency index (EI), which expresses the avidity of antitoxins, is an important instrument for recognizing BoNT subtypes.     

Composition and abundance of resident marsh-surface nekton: comparison between tidal freshwater and salt marshes in Virginia,USA

Previous research on intertidal nekton communities has identifiedimportant determinants of community structure and distribution; however, fewstudies have compared nekton utilization of disparate marsh habitats. Inthis study, abundance and distribution patterns of resident nekton werecompared between tidal freshwater marsh and salt marsh surfaces varying inflooding depth and duration. Nekton were collected in pit traps installedalong elevational transects at four marshes in coastal Virginia (twofreshwater, two saline) from April through November 1992–1993. Thedominant fish collected at all sites was the mummichog Fundulusheteroclitus. The daggerblade grass shrimp Palaemonetes pugio was thedominant nekton species collected at salt marsh sites, and was seasonallyabundant on tidal freshwater marshes. A positive correlation betweenflooding depth and nekton abundance was observed on salt marshes; anopposite pattern was observed on tidal freshwater marshes. Tidal floodingregime influences the abundance of resident nekton, however, the effect maybe confounded by other environmental variables, including variation insurface topography and seasonal presence or absence of submerged aquaticvegetation (SAV) in adjacent subtidal areas. In mid-Atlantic tidalfreshwater wetlands, SAV provides a predation refuge and forage site forearly life stages of marsh-dependent nekton, and several species utilizethis environment extensively. Salt marshes in this region generally lackdense SAV in adjacent subtidal creeks. Consequently, between-sitedifferences in species and size-specific marsh surface utilization byresident nekton were observed. Larvae and juveniles represented 79%and 59% of total fish collected at tidal freshwater and salt marshsites, respectively. The resident nekton communities of tidal freshwater andsalt marsh surfaces are characterized by a few ubiquitous species with broadenvironmental tolerances. This revised version was published online in July 2006 with corrections to the Cover Date.     

Fungal Assemblages Associated with Roots of Halophytic and Non-halophytic Plant Species Vary Differentially Along a Salinity Gradient

Structure of fungal communities is known to be influenced by host plants and environmental conditions. However, in most cases, the dynamics of these variation patterns are poorly understood. In this work, we compared richness, diversity, and composition between assemblages of endophytic and rhizospheric fungi associated to roots of two plants with different lifestyles: the halophyte Inula crithmoides and the non-halophyte I. viscosa (syn. Dittrichia viscosa L.), along a spatially short salinity gradient. Roots and rhizospheric soil from these plants were collected at three points between a salt marsh and a sand dune, and fungi were isolated and characterized by ITS rDNA sequencing. Isolates were classified in a total of 90 operational taxonomic units (OTUs), belonging to 17 fungal orders within Ascomycota and Basidiomycota. Species composition of endophytic and soil communities significantly differed across samples. Endophyte communities of I. crithmoides and I. viscosa were only similar in the intermediate zone between the salt marsh and the dune, and while the latter displayed a single, generalist association of endophytes, I. crithmoides harbored different assemblages along the gradient, adapted to the specific soil conditions. In the lower salt marsh, root assemblages were strongly dominated by a single dark septate sterile fungus, also prevalent in other neighboring salt marshes. Interestingly, although its occurrence was positively correlated to soil salinity, in vitro assays revealed a strong inhibition of its growth by salts. Our results suggest that host lifestyle and soil characteristics have a strong effect on endophytic fungi and that environmental stress may entail tight plant-fungus relationships for adaptation to unfavorable conditions.     

Study of the presence of the spores of Clostridium botulinum in honey in Brazil

The isolation of Clostridium botulinum from honey samples is described. Botulism is characterized as an intoxication provoked by ingestion of contaminated foods with this toxin. Infant botulism happens by the ingestion of spores of C. botulinum together with food that in special conditions of the intestinal tract, such as those present in babies of less than 1 year old, will allow the germination and colonization of the intestine with production and absorption of botulinic toxin. The samples were subjected to dilution and to a thermal shock and cultivated in modified CMM (Difco). Cultures were subjected to Gram smears and toxicity tests in mice. The toxic cultures were purified in RFCA (Oxoid) plates and incubated in anaerobic jars. Positive samples were typed using the mouse assay neutralization test. From the 85 honey samples analyzed, six were positive for C. botulinum (7.06%), and identified as producers of type A, B, and D toxins.     

Osmophilous fungi in the salt marshes of Kuwait.

During an investigation of the mycoflora inhabiting saline soils in Kuwait, special attention was focused on osmophilous fungi. A total of 101 species belonging to 46 genera were encountered from 40 soil samples collected from salt marshes using Czapek's agar supplemented with 40% sucrose. Soil samples were collected from different habitats at different distances from the water edge. Soils near to the water edge were poor in their fungal content, while those taken from areas covered by Juncus arabicus contained highest fungal populations. The recorded genera were classified as follows: 7 were of high frequency of occurrence, 8 moderate, 17 low, and 13 were rare. The order of dominance was Aspergillus, Alternaria, Penicillium, Cephalosporium, Fusarium, Stachybotrys, and Drechslera. Comparison between our results and those in other studies showed that there is no fungal flora characteristic of saline soils.     

Type A and type B botulism in the North: first reported cases due to toxin other than type E in Alaskan Inuit.

Botulism outbreaks shown to be due to type A and type B toxin occurred in Alaska, a region previously known for only type E botulism. The outbreak due to type A toxin involved three people, two of whom died. The outbreak due to type B toxin involved nine people, none of whom died. Both outbreaks were in Inuit villages, and native foods were incriminated. The occurrence of these outbreaks strongly suggests that Clostridium botulinum, types A and B are indigenous to Alaska. The outbreaks underscore the need for initial treatment of patients with antitoxin that is trivalent (ABE), even in Arctic regions.     

Decomposition in estuarine salt marshes: the effect of soil salinity and soil water content

The relation between decomposition rates and soil salinity and moisture conditions in tidal marshes of the Westerschelde estuary was investigated. In the first part of the study, these soil factors were experimentally manipulated in field plots which were either screened from rainwater or which received an additional weekly supply of freshwater from April to September 1989. These treatments had no clear effect on soil salinities and moisture conditions in a low marsh site. Decomposition rates of Spartina anglica leaves (kept in litterbags in the plots) also did not differ between treatments. In screened plots of a middle marsh site, decomposition rate of Elymus pycnanthus leaves decreased significantly. The effect of the experimental treatments on soil moisture content was variable, but comparatively high soil salinity values (up to 61.3) were consistently found in these plots. It is suggested that the elevated salinity levels induced the decrease in decomposition rate.In the second part of the study, cellulolytic decomposition, measured by loss of tensile strength of strips of cotton test cloth, was investigated in relation to a non-manipulated range of soil salinities (3.8–24.2), by exposing the strips in a series of tidal marshes along the salt gradient of the Westerschelde estuary. No correlation between decomposition rate and soil salinity was found. In addition, no relation was found between decomposition rate and soil water content. The results of both parts of this study lead us to the hypothesis that rate limitation of decomposition in estuarine tidal marsh soils is found at high soil salinities only.     

Role of biotic interactions and physical factors in determining the distribution of marsh species along an estuarine salinity gradient

Understanding the mechanisms that shape plant distribution patterns is a major goal in ecology. We investigated the role of biotic interactions (competition and facilitation) and abiotic factors in creating horizontal plant zonation along salinity gradients in the Elbe estuary. We conducted reciprocal transplant experiments with four dominant species from salt and tidal freshwater marshes at two tidal elevations. Ten individuals of each species were transplanted as sods to the opposing marsh type and within their native marsh (two sites each). Transplants were placed at the centre of 9‐m² plots along a line parallel to the river bank. In order to disentangle abiotic and biotic influences, we set up plots with and without neighbouring vegetation, resulting in five replicates per site. Freshwater species (Bolboschoenus maritimus and Phragmites australis) transplanted to salt marshes performed poorly regardless of whether neighbouring vegetation was present or not, although 50–70% of the transplants did survive. Growth of Phragmites transplants was impaired also by competition in freshwater marshes. Salt marsh species (Spartina anglica and Puccinellia maritima) had extremely low biomass when transplanted to freshwater marshes and 80–100% died in the presence of neighbours. Without neighbours, biomass of salt marsh species in freshwater marshes was similar to or higher than that in salt marshes. Our results indicate that salt marsh species are precluded from freshwater marshes by competition, whereas freshwater species are excluded from salt marshes by physical stress. Thus, our study provides the first experimental evidence from a European estuary for the general theory that species boundaries along environmental gradients are determined by physical factors towards the harsh end and by competitive ability towards the benign end of the gradient. We generally found no significant impact of competition in salt marshes, indicating a shift in the importance of competition along the estuarine gradient.     

Using Functional Trajectories to Track Constructed Salt Marsh Development in the Great Bay Estuary, Maine/New Hampshire, U.S.A.

A growing number of studies have assessed the functional equivalency of restored and natural salt marshes. Several of these have explored the use of functional trajectories to track the increase in restored marsh function over time; however, these studies have disagreed as to the usefulness of such models in long‐term predictions of restored marsh development. We compared indicators of four marsh functions (primary production, soil organic matter accumulation, sediment trapping, and maintenance of plant communities) in 6 restored and 11 reference (matched to restored marshes using principal components analysis) salt marshes in the Great Bay Estuary. The restored marshes were all constructed and planted on imported substrate and ranged in age from 1 to 14 years. We used marsh age in a space‐for‐time substitution to track constructed salt marsh development and explore the use of trajectories. A high degree of variability was observed among natural salt marsh sites, displaying the importance of carefully chosen reference sites. As expected, mean values for constructed site (n = 6) and reference site (n = 11) functions were significantly different. Using constructed marsh age as the independent variable and functional indicator values as dependent variables, nonlinear regression analyses produced several ecologically meaningful trajectories (r ²> 0.9), demonstrating that the use of different‐aged marshes can be a viable approach to developing functional trajectories. The trajectories illustrated that although indicators of some functions (primary production, sediment deposition, and plant species richness) may reach natural site values relatively quickly (<10 years), others (soil organic matter content) will take longer.     

Responses of Salt Marsh Ecosystems to Mosquito Control Management Practices along the Atlantic Coast (U.S.A.)

Open marsh water management (OMWM) of salt marshes modifies grid‐ditched marshes by creating permanent ponds and radial ditches in the high marsh that reduce mosquito production and enhance fish predation on mosquitoes. It is preferable to using pesticides to control salt marsh mosquito production and is commonly presented as a restoration or habitat enhancement tool for grid‐ditched salt marshes. Monitoring of nekton, vegetation, groundwater level, soil salinity, and bird communities before and after OMWM at 11 (six treatment and five reference sites) Atlantic Coast (U.S.A.) salt marshes revealed high variability within and among differing OMWM techniques (ditch‐plugging, reengineering of sill ditches, and the creation of ponds and radial ditches). At three marshes, the dominant nekton shifted from fish (primarily Fundulidae species) to shrimp (Palaemonidae species) after manipulations and shrimp density increased at other treatment sites. Vegetation changed at only two sites, one with construction equipment impacts (not desired) and one with a decrease in woody vegetation along existing ditches (desired). One marsh had lower groundwater level and soil salinity, and bird use, although variable, was often unrelated to OMWM manipulations. The potential effects of OMWM manipulations on non‐target salt marsh resources need to be carefully considered by resource planners when managing marshes for mosquito control.     

Aquatic insect emergence in two Great Lakes marshes

This study determined total number, biomass, taxa, and seasonal occurrence of adult aquatic insects emerging from four vegetation zones in one diked and one undiked freshwater coastal marsh on hypereutrophic lower Green Bay, Lake Michigan, USA during the summer of 1984. Floating box traps were placed in open water, sparse emergent, dense emergent, and wet meadow vegetation zones in each marsh. Insects were collected during 20 24-hour periods, each four days apart, from June 11 to August 26. Two-way ANOVA was used to test differences in number and biomass of insects between marshes and among vegetation zones. Polynomial regression was used to evaluate seasonal emergence patterns. More insects, insect biomass, and insect taxa were found in the diked marsh, especially during the first half of the sampling period. Damselflies were much more abundant in the diked marsh. Most insects and insect biomass were found in the sparse emergent vegetation zone of both marshes. The emerging insect community in the diked marsh appears enhanced by its separation from the hypereutrophic and turbid waters of lower Green Bay.     

Keratinolytic fungi in Egyptian soils

The frequency of occurrence of keratinolytic fungi in seventy soil samples collected from different sites in Upper Egypt and in the coastal area of the Mediterranean and baited with human and animal hair and pigeon feathers was determined.Twenty-one species in addition to an unidentified species, which belong to sixteen genera were collected.Chrysosporium indicum, C. tropicum, C. keratinophilum andMicrosporum gypseum were the most frequent fungal species recovered from baited soils.The eight soil samples collected from the salt marshes of the coastal Mediterranean area were completely free from any keratinolytic fungi, whereas the sixty-two soil samples collected from cultivated soils contributed a species ranging from one to four species.     

Acute toxicity of aminoglycoside antibiotics as an aid in detecting botulism.

Gentamicin sulfate or neomycin sulfate injected intraperitoneally into 24- to 27-g mice at a dose of 6.2 mg per mouse elicited botulism-like responses in less than 30 min, but a dose of 3.1 mg per mouse had no observable effect. The normally nontoxic 3.1-mg aminoglycoside dose aggravated the illness induced by an earlier injection of Clostridium botulinum type A or B toxin; it was usually lethal in 2 to 20 min if the preexisting illness was moderate to severe and worsened the condition of mice for about 30 min if the preexisting botulism was mild. The aminoglycoside had no effect when given shortly after the botulinum toxin was injected intraperitoneally; the sensitized state followed a latent period. It rapidly produced botulism-like effects when given to mice which had responded to a mixture of botulinum toxin and another mouse toxic agent with an illness that did not include signs of botulism. An unexpected illness devoid of botulism-like effects was encountered during intestinal colonization of mice by C. botulinum. The appearance of botulism-like signs soon after 3.1 mg of gentamicin sulfate was injected supported other suggestions that this illness included botulism that was masked by the effects of a second cause.     

荒漠草原盐沼湿地苦豆子土壤细菌群落构建机制及其影响因素

【背景】荒漠草原盐沼湿地是陆地生态系统的重要组成部分,土壤水分和盐分变化是影响该生态系统土壤细菌群落构建的重要因素。【目的】土壤细菌群落构建是由确定性和随机性主导的连续生态过程,阐明荒漠草原盐沼湿地土壤细菌群落的构建机制对于加深微生物作为关键生态系统因子重要性的理解具有积极意义。【方法】以宁夏中部典型荒漠草原盐沼苦水湖湿地为研究对象,对近湖边（near the lake,NL）和远离湖边（far from the lake,FL）苦豆子群落土壤理化特性进行测定并结合土壤细菌高通量测序分析。【结果】NL和FL样地具有明显的水盐梯度变化,NL样地土壤pH、含水量和电导率均显著高于FL样地;变形菌门、放线菌门、厚壁菌门、拟杆菌门和黏菌门是研究区域土壤细菌群落的优势菌门,变形菌门相对丰度随水盐梯度上升而升高,放线菌门和厚壁菌门相对丰度则随之下降,门下成员大多与水盐变化具有明显的相关性;此外,FL样地土壤细菌网络则具有稳定的网络关系;随着NL样地向FL样地的延伸,土壤细菌群落由随机过程主导,并且受pH、电导率和环境变量的影响。【结论】荒漠草原盐沼湿地水分和盐分的变化改变了土壤细菌群落结构;土壤细菌群落通过生态位占据等策略提高逆境下的生存能力;细菌群落构建是随机过程和确定性过程组成的连续统一体,同样受环境变化的影响。本结果揭示了荒漠草原盐沼湿地细菌群落结构和相互关系对环境变化的响应特征,同时阐明了该区土壤细菌群落的构建机制及影响因素,也为相关科学研究提供了一定理论参考。     

Consolidation and volumetric soil-water content of salt marsh soils following habitat modification for mosquito control

The runnelling form of habitat modification for mosquito control in saltmarsh increases tidal frequency, and may affect soil properties such as volumetric soil-water content and consolidation. The effects of habitat modification on soil properties are in turn likely to affect ecological processes. Runnels constructed mechanically to a depth of no more than 0.3 m with smooth, spoon shaped edges linked isolated mosquito-breeding pools in the high marsh to the tidal source at the saltmarsh/mangrove interface. The physical design of runnels may result in a significant increase in the frequency of flooding tidal events that flush isolated mosquito-breeding pools. Impacts of the runnelling technique were determined at three marshes using two sampling protocols: (a) comparisons between modified and unmodified shores and (b) comparisons with lateral distance from a runnel. At one marsh, volumetric water content was significantly higher at runnelled than at unrunnelled sites after tides that only partly inundated the marsh, but this pattern was not found at the other marshes. Soil consolidation was greater further from the shore, but was not different between runnelled and unrunnelled shores. Measurements at different lateral distances from runnels demonstrated higher water content levels and lower consolidation up to 5 m from runnels and no effect further away. The varied responses to runnelling at different marshes may reflect specific site characteristics such as slope and hydraulic tidal forces. Remedial strategies for similar mosquito control techniques, based on habitat modification, should include dynamic classifications of saltmarshes.