Reference values for acetyl and butyrylcholinesterases in cattle under actual management conditions, hepatic and renal function by application of chlorpyrifos.

Chlorpyrifos is an anticholinesterase organophosphate insecticide widely used in Argentina in the production of food derived from animal, fruit and horticultural origin and is reported as a residue within these products. Local reference values for acetyl and butyrylcholinesterase were determined in Aberdeen Angus bovine and cross bred cattle (n = 25), a requirement to be able to evaluate toxicity of commercial organophosphate and carbamate formulations. The activity of cholinesterase enzymes presented an overall mean of 2,183.00 ± 485.6 IU L-1 for erythrocyte acetylcholinesterase and 203.1 ± 42.06 IU L-1 for plasma butyrylcholinesterase, which are used as reference values for meat steers within a system of intensive production in a semi-arid region. The toxic potential of chlorpyrifos in steers of the same breeds (n = 12) was assessed applying chlorpyrifos 15.00% Tipertox® in a single therapeutic dose of 7.50 mg kg-1 by topical route. Prior to application and then on day 1 and day 21 post-application, both blood cholinesterases, serum chlorpyrifos concentration by ultra-high resolution liquid chromatography with mass detector, analysis of blood counts, total proteins, liver enzymes, urea and creatinine were evaluated. The mean plasma concentration of chlorpyrifos was 27.90 ug L-1 at 24 h. The findings indicate that the therapeutic treatment of castrated male bovines treated with chlorpyrifos, applied by pour-on according to the manufacturer's instructions, does not cause changes in the variables evaluated.

Prokaryotic Community in Lacustrine Sediments of Byers Peninsula (Livingston Island, Maritime Antarctica).

Byers Peninsula (Livingston Island, Antarctica), the largest seasonally ice-free region of the Maritime Antarctica, holds a large number of lakes, ponds, and streams. The prokaryotic structure and bacterial diversity in sediment samples collected during the 2008-2009 austral summer from five inland lakes, two coastal lakes, and an estuarine site were analyzed by Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (CARD-FISH) and 16S rRNA 454 tag pyrosequencing techniques, respectively. Differently from inland lakes, which range around the oligotrophic status, coastal lakes are eutrophic environments, enriched by nutrient inputs from marine animals. Although the prokaryotic abundances (estimated as DAPI stained cells) in sediment samples were quite similar among inland and coastal lakes, Bacteria always far dominated over Archaea. Despite the phylogenetic analysis indicated that most of sequences were affiliated to a few taxonomic groups, mainly referred to Proteobacteria, Bacteroidetes, and Actinobacteria, their relative abundances greatly differed from each site. Differences in bacterial composition showed that lacustrine sediments were more phyla rich than the estuarine sediment. Proteobacterial classes in lacustrine samples were dominated by Betaproteobacteria (followed by Alphaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria), while in the estuarine sample, they were mainly related to Gammaproteobacteria (followed by Deltaproteobacteria, Epsilonproteobacteria, Alphaproteobacteria, and Betaproteobacteria). Higher number of sequences of Alphaproteobacteria, Cyanobacteria, Verrucomicrobia, and Planctomycetes were observed in sediments of inland lakes compared to those of coastal lakes, whereas Chloroflexi were relatively more abundant in the sediments of coastal eutrophic lakes. As demonstrated by the great number of dominant bacterial genera, bacterial diversity was higher in the sediments of inland lakes than that in coastal lakes. Ilumatobacter (Actinobacteria), Gp16 (Acidobacteria), and Gemmatimonas (Gemmatimonadetes) were recovered as dominant genera in both inland and coastal lakes, but not in the estuarine sample, indicating that they may be useful markers of Antarctic lakes. The proximity to the sea, the different lake depths and the external or internal origin of the nutrient sources shape the bacterial communities composition in lacustrine sediments of Byers Peninsula.

Changes in prokaryotic community composition accompanying a pronounced temperature shift of a shallow marine thermal brine pool (Panarea Island, Italy).

Hot Lake is a recently described thermal brine pool off Panarea Island (Eolian Islands, Italy) where emitted fluids are highly saline and rich in CO2 and H2S. The prokaryotic community composition in surface sediment samples was analyzed by high-throughput Illumina sequencing targeting the V3 region of the 16S rRNA at two time points that differed mainly with respect to temperature conditions, high-temperature (94 °C, HT09) and low-temperature (28.5 °C, LT10). Bacterial richness and diversity were greater than those of Archaea under both temperature conditions. In contrast to Bacteria, diversity and evenness of Archaea greatly increased at LT10. While the phylogenetic analysis indicated the presence of members mostly affiliated with the same taxonomic groups, their relative abundances differed from HT09 to LT10, resulting in different bacterial and archaeal assemblages. Both HT09 and LT10 were dominated by members of the Epsilonproteobacteria. Within this subphylum, bacteria of the genus Sulfurimonas were most frequently detected at HT09, while Arcobacter prevailed at LT10. The abundance of other dominant taxonomic groups (≥1 % of Illumina reads) also correlated with temperature conditions. Members assigned to hyperthermophilic Euryarchaeota (Thermococci) or to thermophilic (Caldiserica) and thermoresistant (Firmicutes) bacterial taxa were dominant at HT09, while those related to non-thermophilic Bacteroidetes, Fusobacteria and Actinobacteria were dominant at LT10. Several, probably photosynthetic, members of the Alphaproteobacteria, Chlorobi, Cyanobacteria and Chloroflexi were recovered under both temperature conditions. The co-occurrence of photosynthetic and chemolithotrophic microorganisms represents a unique feature of shallow vents such as Hot Lake.

Diversity of prokaryotic community at a shallow marine hydrothermal site elucidated by Illumina sequencing technology.

To investigate the prokaryotic community structure and composition in an active hydrothermal site, named Black Point, off Panarea Island (Eolian Islands, Italy), we examined sediment and fluid samples, differing in temperature, by a massive parallel sequencing (Illumina) technique targeting the V3 region of the 16S rRNA gene. The used technique enabled us to detect a greater prokaryotic diversity than that until now observed and to reveal also microorganisms occurring at very low abundance (≤0.01 %). Most of sequences were assigned to Bacteria while Archaea were a minor component of the microbial community in both low- and high-temperature samples. Proteobacteria (mainly consisting of Alpha-, Gamma-, and Epsilonproteobacteria) dominated among all samples followed by Actinobacteria and Bacteroidetes. Analyzed DNA obtained from samples taken at different temperatures indicated the presence of members of different dominant genera. The main differences were observed between sediment samples where Rhodovulum and Thiohalospira prevailed at high temperature, while Thalassomonas and Sulfurimonas at low temperature. Chlorobium, Acinetobacter, Sulfurimonas, and Brevundimonas were abundant in both low- and high-temperature fluid samples. Euryarchaeota dominated the archaeal community in all samples. Classes of Euryarchaeota embracing hyperthermophilic members (Thermococci and Thermoplasmata) and of Crenarchaeota (Thermoprotei) were more abundant in high-temperature samples. A great number of sequences referred to Bacteria and Archaea still remained unaffiliated, indicating that Black Point site represents a rich source of so-far uncharted prokaryotic diversity.

A novel EPS-producing strain of Bacillus licheniformis isolated from a shallow vent off Panarea Island (Italy).

A haloalkaliphilic, thermophilic Bacillus strain (T14), isolated from a shallow hydrothermal vent of Panarea Island (Italy), produced a new exopolysaccharide (EPS). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain T14 was highly related (99 % similarity) to Bacillus licheniformis DSM 13(T) and Bacillus sonorensis DSM 13779(T). Further DNA-DNA hybridization analysis revealed 79.40 % similarity with B. licheniformis DSM 13(T) and 39.12 % with B. sonorensis DSM 13779(T). Sucrose (5 %) was the most efficient carbon source for growth and EPS production. The highest EPS production (366 mg l(-1)) was yielded in fermenter culture at 300 rpm after 48 h of incubation. The purified fraction EPS1 contained fructose/fucose/glucose/galactosamine/mannose in a relative proportion of 1.0:0.75:0.28:tr:tr and possessed a molecular weight of 1,000 kDa displaying a trisaccharide unit constituted by sugars with a ß-manno-pyranosidic configuration. Screening for biological activity showed anti-cytotoxic effect of EPS1 against Avarol in brine shrimp test, indicating a potential use in the development of novel drugs.

Vertical distribution of Archaea and Bacteria in a meromictic lake as determined by fluorescent in situ hybridization.

The prokaryotic cells distribution in the water column of the coastal saline meromictic Lake Faro (Messina, Italy) was investigated by microscopic counting techniques. Water samples were collected at a central station from the surface to the bottom, when waters were characterized by a marked stratification. A "red-water" layer, caused by a dense growth of photosynthetic sulfur bacteria, was present at a depth of 15 m, defining a transition area between oxic (mixolimnion) and anoxic (monimolimnion) layers. Fluorescently labeled 16S rRNA oligonucleotide, group-specific probes were used to determine the abundance of Bacteria and Archaea, and their subgroups, Green Sulfur Bacteria (GSB), Sulfate Reducing Bacteria (SRB), Cyanobacteria and Chromatium okenii, and Crenarchaeota and Euryarchaeota, as key elements of the microbial community. Bacteria decreased from surface to bottom, while Archaea increased with depth and reached the maximum value at 30 m, where they outnumbered the Bacteria. Bacteria and picophytoplankton prevailed in the mixolimnion. At the chemocline high numbers of prokaryotic cells were present, mainly represented by Cyanobacteria, Chromatium okenii and Euryarchaeota. GSB, SRB, and Crenarchaeota prevailed below the chemocline. Although Archaea constitute a minor fraction of microbial community, they could represent active contributors to the meromictic Lake Faro ecosystem.

Distribution and diversity of bacteria in a saline meromictic lake as determined by PCR-DGGE of 16S rRNA gene fragments.

The variations in vertical distribution and composition of bacteria in the meromictic Lake Faro (Messina, Italy) were analysed by culture-independent methods in two different mixing conditions. Water samples were collected from a central station from the surface to the bottom (30 m depth) on two different sampling dates--the first characterised by a well-mixed water mass and the second by a marked stratification. A 'red-water' layer, caused by a dense growth of photosynthetic sulphur bacteria, was present at a depth of 25 m in December 2005 and at 15 m in August 2006, defining two different zones in terms of their physicochemical properties. The vertical distribution of bacterioplankton showed that the interface zones were more densely populated than others. In both sampling periods, the highest numbers of live cells were observed within 'red water' layers. The dominant phylotypes of the bacterial community were determined by sequencing the Denaturing Gradient Gel Electrophoresis (DGGE) bands resulting from PCR amplification of 16S rRNA gene fragments. The number of DGGE bands, considered indicative of the total species richness, did not vary predictably across the two different sampling periods. Proteobacteria (α-, γ-, δ- and ε subclass members), Cytophaga-Flavobacterium-Bacteroides, green sulphur bacteria and Cyanobacteria were retrieved from Lake Faro. Most of the bands showed DNA sequences that did not match with other previously described organisms, suggesting the presence of new indigenous bacterial phylotypes.

High prevalence of arcobacter carriage in older subjects with type 2 diabetes.

Arcobacters are potential pathogens related to diarrheic infections and, rarely, septicaemia. This study evaluated the prevalence of arcobacters in stool samples of subjects with (n = 38) and without (n = 61) type 2 diabetes by using cultural and molecular techniques. Three Arcobacter positive cultures were found, all among diabetic subjects, whereas molecular analysis showed a carriage rate of 79% and 26.2% in subjects with and without type 2 diabetes (P < .001), respectively. The multivariate analysis showed that type 2 diabetes (beta = 1.913; 95%CI: 2.378-19.285; P < .0001) and age (beta = 1.744; 95%CI: 2.077-15.766; P = .001) were the only factors independently associated with arcobacters colonization in this population. Our study demonstrated a high prevalence of arcobacters colonization in type 2 diabetic and older subjects. The clinical significance and the potential health risk associated with these emerging species remain to be determined.

Biomonitoring of the hematological, biochemical and genotoxic effects of the mixture cypermethrin plus chlorpyrifos applications in bovines.

External antiparasitic agents applied in bovine production represent a risk to consumers of meat products, especially if the conditions of their use are not strictly respected. The post-mortem control of residues in meat is an activity that must be updated and reinforced by the biomonitoring of live animals and the use of analytical tools to help identifying signs of early warning risks. The objective of the present study was to carry out a pre-slaughter biomonitoring approach in Aberdeen Angus cattle and crosses (n = 12) with the application of a commercial formulation of cypermethrin plus chlorpyrifos. This was performed with a single therapeutic dose applied on the backs of the cattle, through hematological, enzymatic, as well as hepatic and renal function analysis in plasma, and then quantifying the genotoxic effect on lymphocytes. Analytical measurements of plasma concentrations of cypermethrin plus chlorpyrifos at 24 h were negative and therefore a low absorption of the compounds was assumed. Measurement of acetyl cholinesterase showed no inhibition after exposure. The concentration of urea increased between 24 h and 168 h post application of the formulation, without showing any kidney damage. The rest of the parameters analyzed did not show any variations. This evaluation of hematological and biochemical effects and of cytokinesis-block micronucleus cytome assay in bovines is proposed as a pre-slaughter control of biomonitoring of the health status of animals, with a focus on food safety for meat consumers.

Bacterial and archaeal populations at two shallow hydrothermal vents off Panarea Island (Eolian Islands, Italy).

The aim of this study was to investigate the microbial community thriving at two shallow hydrothermal vents off Panarea Island (Italy). Physico-chemical characteristics of thermal waters were examined in order to establish the effect of the vents on biodiversity of both Bacteria and Archaea. Water and adjacent sediment samples were collected at different times from two vents, characterised by different depth and temperature, and analysed to evaluate total microbial abundances, sulphur-oxidising and thermophilic aerobic bacteria. Total microbial abundances were on average of the order of 10(5) cells ml(-1), expressed as picoplanktonic size fraction. Picophytoplanktonic cells accounted for 0.77-3.83% of the total picoplanktonic cells. The contribution of bacterial and archaeal taxa to prokaryotic community diversity was investigated by PCR-DGGE fingerprinting method. The number of bands derived from bacterial DNA was highest in the DGGE profiles of water sample from the warmest and deepest site (site 2). In contrast, archaeal richness was highest in the water of the coldest and shallowest site (site 1). Sulphur-oxidising bacteria were detected by both culture-dependent and -independent methods. The primary production at the shallow hydrothermal system of Panarea is supported by a complex microbial community composed by phototrophs and chemolithotrophs.

Water quality and ecological status of the Alcantara River estuary (Italy).

The Alcantara River estuary was studied to obtain the first data on both water quality and ecological status of the only River Park in Sicily (Italy). Water samples were seasonally collected from three selected stations in the estuarine area and from one station at the mouth of the estuary in the marine coastal zone. Picoplankton and picophytoplankton counts were among the highest observed in estuarine environments and in other coastal waters of the Mediterranean Sea. The contribution of picophytoplankton to total picoplankton showed greater variations in freshwater (from 0.53% to 8.6%) than in coastal waters (from 1.8% to 4.3%). Picophytoplankton abundance increased in Alcantara waters in winter. Vibrio and Aeromonas spp. abundances ranged from x10(2) to x10(3) CFU 100 ml(-1) and from x10(3) to x10(5) CFU 100 ml(-1) respectively. Vibrio and Aeromonas spp. counts were independent from the fecal contamination level. Bacterial identification of the isolates revealed the presence of pathogenic and potentially pathogenic Vibrio and Aeromonas spp. for humans and animals.

Detection and enumeration of Arcobacter spp. in the coastal environment of the Straits of Messina (Italy).

Seawater and plankton samples from the Straits of Messina, Italy, were analysed to confirm the occurrence of potentially pathogenic Arcobacter butzleri, A. cryaerophilus and A. skirrowii. Both classical cultural methods and molecular techniques were used as confirmative steps of the growth in enrichment broth to enumerate and differentiate these bacteria. Only A. butzleri was isolated from seawater and plankton samples and was more abundant when associated with plankton than free-living. A. cryaerophilus was occasionally detected by PCR assay from environmental samples. The PCR procedure, used in a combined method, was useful in enumerating Arcobacter spp. in marine environment.