Abstract: Listeriolysin O (LLO) is the major factor implicated in the escape of Listeria monocytogenes from the phagolysosome. It is the only representative of cholesterol-dependent cytolysins that exhibits pH-dependent activity. Despite intense studies of LLO pH-dependence, this feature of the toxin still remains incompletely explained. Here we used fluorescence and CD spectroscopy to show that the structure of LLO is not detectably affected by pH at room temperature. We observed slightly altered haemolytic and permeabilizing activities at different pH values, which we relate to reduced binding of LLO to the lipid membranes. However, alkaline pH and elevated temperatures caused rapid denaturation of LLO. Aggregates of the toxin were able to bind Congo red and Thioflavin T dyes and were visible under transmission electron microscopy as large, amorphous, micrometer-sized assemblies. The aggregates had the biophysical properties of amyloid. Analytical ultracentrifugation indicated dimerization of the protein in acidic conditions, which protects the protein against premature denaturation in the phagolysosome, where toxin activity takes place. We therefore suggest that LLO spontaneously aggregates at the neutral pH found in the host cell cytosol and that this is a major mechanism of LLO inactivation.
Abstract: Male glandular organs characterized by porous surfaces with hair-like cuticular elaborations are known from several trichoniscid isopods. In the subterranean species Titanethes albus, males possess paired tubercles with numerous hairs and pores dorsally on the pleon. We analyzed the microscopic anatomy of these structures with scanning and transmission electron microscopy. Diverse epicuticular formations and numerous sensilla, which are probably chemoreceptive, are present on the tubercles. We found several secretory surfaces on the pleon in addition to the dorsal tubercles. We also examined the distribution, architecture and ultrastructure of male-specific glands in T.albus with light and transmission electron microscopy. Three distinct types of male-specific rosette glands are present in different parts of the pleon and in the uropods. Glands secreting on the dorsal tubercles contain stellar central cells. The ultrastructure and histochemical staining properties of male-specific glands in T. albus suggest that they produce peptides which might function as contact pheromones.
Abstract: To examine effects of various environmental factors on adsorption and inactivation of Pseudomonas aeruginosa-specific phages: δ (family Podoviridae), J-1, Ï-1 and 001A (family Siphoviridae) and their ability to inhibit bacterial growth and biofilm formation.
Abstract: Because endogenous cellulases have been observed in arthropods, the potential ability to produce cellulose degrading enzymes was examined in the terrestrial isopod Porcellio scaber, an important decomposer of decayed plant material. cDNA fragments encoding portions of two novel endo-β-1,4-glucanase amino acid sequences were amplified by RT-PCR, and the amino acid sequences predicted were affiliated to endo-β-1,4-glucanases from other arthropods, where they cluster with endo-β-1,4-glucanases of decapod crustaceans. Hybridization in situ reveals the hepatopancreas to be the primary site of gene expression and provides direct evidence of the endogenous origin of endo-β-1,4-glucanase in P. scaber. Conservation of catalytically important amino acid residues suggests that both sequences translate into functional cellulases. Cellulolytic activity was detected in hepatopancreatic extract after separation by SDS-PAGE, which included CMC as substrate. This is the first evidence of endogenous cellulases in peracarid crustaceans and gives strong support for the involvement of isopod endo-β-1,4-glucanases in the degradation of cellulose in their diet.
Abstract: The aim of the study was to screen various kinds of samples for Pseudomonas aeruginosa specific phages and to isolate and partially characterize those with broad activity spectra. The Pseudomonas specific phages were isolated using an enrichment procedure with single strains or the cocktail of P. aeruginosa strains as hosts. Using the described procedure, phages were successfully isolated only from water samples, while in soil and feces no Pseudomonas specific phages were detected. The lytic spectra of isolated phages were determined by spot method on lawns of 33 P. aeruginosa strains and five species belonging to family Enterobacteriaceae. The results showed that among isolated phages, 001A, delta, and I possessed the broad activity spectra, as were able to plaque on more than 50% of tested P. aeruginosa strains, while none of the phages were able to lyse the other tested species. Significant differences in phage activity spectra were not observed when P. aeruginosa cocktail was applied for sample enrichment. The most of the phages examined by electron microscopy belonged to family Siphoviridae, while the broad activity spectra isolates, except for 001A, possessed morphological characteristics of family Podoviridae. Digested DNA of the phages delta and I showed similar patterns, indicating the prevalence and success of this phage type in the environment.
Abstract: New products of nanotechnologies, including nanoparticles, need to be assessed according to their biological reactivity and toxic potential. Given the large number of diverse nanomaterials, a tiered approach is favoured. The aim of our work presented here is to elaborate an in vivo assay with terrestrial invertebrates (Porcellio scaber), which could serve as a first step of hazard identification of nanoparticles. We adapted the widely used acridine orange/ethidium bromide (AO/EB) assay to be applicable for cell membrane stability assessment of entire organ where the animal was exposed in vivo. The digestive glands (hepatopancreas) of terrestrial isopods were taken as a model test system. The assay was validated with Cu(NO(3))(2) and surfactants. The results showed that all tested nanoparticles, i.e. nanosized TiO(2), nanosized ZnO and fullerenes (C(60)) have cell membrane destabilization potential. As expected, C(60) is the most biologically potent. The AO/EB in vivo assay proved to be fast because response is recorded after 30 min of exposure, relatively simple because digestive glands are inspected immediately after isolation from exposed animals and promising approach because different types of nanoparticles could be tested for their biological potential. This assay provides data for the identification of hazardous potential of nanoparticles before subsequent steps in a tiered approach are decided.
Abstract: We report the use of a focused ion beam/scanning electron microscope (FIB/SEM) for simultaneous investigation of digestive gland epithelium gross morphology and ultrastructure of multilamellar intracellular structures. Digestive glands of a terrestrial isopod (Porcellio scaber, Isopoda, Crustacea) were examined by FIB/SEM and by transmission electron microscopy (TEM). The results obtained by FIB/SEM and by TEM are comparable and complementary. The FIB/SEM shows the same ultrastructural complexity of multilamellar intracellular structures as indicated by TEM. The term lamellar bodies was used for the multillamellar structures in the digestive glands of P. scaber due to their structural similarity to the lamellar bodies found in vertebrate lungs. Lamellar bodies in digestive glands of different animals vary in their abundance, and number as well as the thickness of concentric lamellae per lamellar body. FIB/SEM revealed a connection between digestive gland gross morphological features and the structure of lamellar bodies. Serial slicing and imaging of cells enables easy identification of the contact between a lamellar body and a lipid droplet. There are frequent reports of multilamellar intracellular structures in different vertebrate as well as invertebrate cells, but laminated cellular structures are still poorly known. The FIB/SEM can significantly contribute to the structural knowledge and is always recommended when a link between gross morphology and ultrastructure is investigated, especially when cells or cellular inclusions have a dynamic nature due to normal, stressed or pathological conditions.
Abstract: Pointed, rod-shaped bacteria colonizing the cuticular surface of the hindgut of the terrestrial isopod crustacean Porcellio scaber (Crustacea: Isopoda) were investigated by comparative 16S rRNA gene sequence analysis and electron microscopy. The results of phylogenetic analysis, and the absence of a cell wall, affiliated these bacteria with the class Mollicutes, within which they represent a novel and deeply branched lineage, sharing less than 82.6% sequence similarity to known Mollicutes. The lineage has been positioned as a sister group to the clade comprising the Spiroplasma group, the Mycoplasma pneumoniae group, and the Mycoplasma hominis group. The specific signature sequence was identified and used as a probe in in situ hybridization, which confirmed that the retrieved sequences originate from the attached rod-shaped bacteria from the hindgut of P. scaber and made it possible to detect these bacteria in their natural environment. Scanning and transmission electron microscopy revealed a spherically shaped structure at the tapered end of the rod-shaped bacteria, enabling their specific and exclusive attachment to the tip of the cuticular spines on the inner surface of the gut. Specific adaptation to the gut environment, as well as phylogenetic positioning, indicate the long-term association and probable coevolution of the bacteria and the host. Taking into account their pointed, rod-shaped morphology and their phylogenetic position, the name "Candidatus Bacilloplasma" has been proposed for this new lineage of bacteria specifically associated with the gut surface of P. scaber.
Abstract: SUMMARY Clinicopathological and electron microscopical findings of eight cases of enzootic nasal adenocarcinoma of sheep, diagnosed solely in one big flock in Slovenia between years 2001 and 2003 are described. All affected sheep were female, their mean age was 4.5 +/- 1.5 years and they either belonged to the Istrian pramenka breed (five sheep) or were crossbreeds (three sheep). Tumours that arose from the ethmoid area of the nasal cavity were unilateral in six cases (75%) and bilateral in two cases (25%). All tumours were classified as adenocarcinomas by histopathological examination and they displayed either a combination of tubular and papillary growth or less often solely tubular proliferation. No metastases were detected in regional lymph nodes, brain or other organs. Electron microscopical studies performed on the reprocessed paraffin-embedded tissues revealed the presence of the virus-like particles with an average diameter between 70 and 90 nm.
Abstract: Lake Skadar is the largest lake of the Balkan Peninsula, located along the Montenegro-Albanian border. The unique features of the lake and wide range of endemic, and rare or endangered plant and animal species, resulted in the classification of the Skadar as a wetland site of international significance. In spite of its importance, the lake is influenced by inflowing waters from the river Moraca and other regional rivers contaminated by the industry, municipal and agricultural activities in the area. Therefore, the lake has been the subject of various physical, chemical, biological and toxicological examinations. However, community-level analyses are most relevant to assess the effect of stressors on aquatic ecosystems. In the present study, bacterial community structure among differently polluted sites of the lake was compared using a genetic fingerprinting technique.
Abstract: Intracellular bacteria were observed in the hepatopancreas of the terrestrial isopod Porcellio scaber. Comparative 16S rRNA gene sequence analysis and electron microscopic observations were used to determine the taxonomic position of these intracellular bacteria. Phylogenetic analysis and a complex developmental cycle affiliate these bacteria to the order Chlamydiales, within which they form a distinctive lineage, close to the family Simkaniaceae. They share <92 % 16S rRNA gene sequence similarity with their closest relative and <88 % similarity with other members of the order Chlamydiales. A specific signature oligonucleotide sequence was identified and used as a probe, enabling the identification of intracellular bacteria in infected hepatopancreatic tissue. According to the distinctive morphology of their elementary bodies, which are rod-shaped rather than spherical and contain translucent oblong structures, their genomic properties and their crustacean host, the name 'Candidatus Rhabdochlamydia porcellionis' is proposed for intracellular bacteria in the hepatopancreas of P. scaber.
Abstract: Anaerobic bacteria from Porcellio scaber hindgut were identified and, subsequently, isolated using molecular approach. Phylogenetic affiliation of bacteria associated with the hindgut wall was determined by analysis of bacterial 16S rRNA gene sequences which were retrieved directly from washed hindguts of P. scaber. Sequences from bacteria related to obligate anaerobic bacteria from genera Bacteroides and Enterococcus were retrieved, as well as sequences from 'A1 subcluster' of the wall-less mollicutes. Bacteria from the genus Desulfotomaculum were isolated from gut wall and cultivated under anaerobic conditions. In contrast to previous reports which suggested the absence of anaerobic bacteria in the isopod digestive system due to short retention time of the food in the tube-like hindgut, frequent renewal of the gut cuticle during the moulting process, and unsuccessful attempts to isolate anaerobic bacteria from this environment our results indicate the presence of resident anaerobic bacteria in the gut of P. scaber, in spite of apparently unsuitable, i.e. predominantly oxic, conditions.
Abstract: Rumen bacterium Pseudobutyrivibrio xylanivorans strain Mz5T possessed a potent xylanolytic enzyme system consisting of at least 7 different xylan hydrolases with molar mass 27-145 kDa. Three of them were successfully isolated in active native form. This strain produced butyrate and lactate on different saccharides. cis-9, trans-11-Conjugated linoleic acid was also detected in the culture medium. Bacteriocin-like inhibitory substances of Mz5T were active against some strains of rumen bacteria and against selected Salmonella and E. coli isolates from poultry meat. The strain Mz5T retained viability and xylanolytic activity also under not fully anaerobic conditions; its cells attached to the Caco-2 cells so that its successful association with gut epithelial cells may be expected. These in vitro results confirmed several probiotic traits of the isolate Mz5T and justified further in vivo experiments to test its ability to improve animal health and performance.
Abstract: Recently developed molecular biology approaches make possible the detailed genetic, taxonomic and ecological examination of microorganisms from various habitats. Animal gut represents one of the most complex microbial ecosystems with a large degree of microbial biodiversity present. Bacteria inhabiting the gut usually play important roles in metabolic transformations of substrates and sometimes, e.g. in ruminants, they make the basis for an obligate symbiosis with the host. Here we discuss molecular microbiology as a strategy for examination of gut bacteria, concentrating on a typical and in such environment dominant group of strictly anaerobic Gram-negative bacteria from the phylogenetic group Cytophaga/Flexibacter/Bacteroides. The bacteria from the genus Prevotella are the most abundant Gram-negative bacteria in the rumen and form a distinctive phylogenetic cluster, clearly separated from prevotellas isolated from other ecological niches. They may represent a good choice for a model organism in genetic manipulation experiments and for studies of gene transfer mechanisms taking place in the gut. The molecular tools for detection and monitoring of ruminal prevotellas are discussed.
Abstract: Molecular approaches were used to examine the genetic diversity of bacteria associated with the gut wall of the terrestrial isopod Porcellio scaber and to determine whether an autochthonous microflora exists in the P. scaber hindgut. 16S ribosomal genes were amplified from the total DNA isolated from thoroughly washed papillate regions of the hindgut, where the highest concentrations of bacteria are commonly found. The amplified genes were cloned, sequenced and phylogenetically analysed. The results implied an unexpectedly large diversity of microflora associated with the cuticle of the hindgut. Almost half of the retrieved sequences were found to be less than 80% homologous with any of the known sequences available at DNA data banks. Most of these sequences were clustered in one of three groups, and were clearly distant from the sequences of other bacterial taxa, indicating that they could represent novel bacterial species or even genera. More than two thirds of the sequences were found to be phylogenetically related to sequences from bacteria typically isolated from human and animal intestines, e.g. streptococci, enterococci, and members of the genus Bacteroides. The majority of the remaining sequences were most closely related to typical soil bacteria, e.g. bacilli and pseudomonads. The facts that a large proportion of the retrieved sequences was related to the sequences of bacteria, which are autochthonous to intestinal ecosystems, and that bacteria, specifically attached to the cuticular spines, were observed, indicate that truly autochthonous bacteria may well be present in the hindgut of P. scaber.
Abstract: A new spirochetal strain was isolated from the rumen of a black-and-white Holstein cow and preliminarily characterized. The sugar fermentation tests and morphological observations indicated this organism to be a member of a novel, as yet undescribed spirochetal rumen species. The small subunit ribosomal RNA genes were amplified and the PCR products were cut with the restriction endonucleases TaqI, DdeI, HhaI and Sau3AI. The comparison of the observed RFLP with the hypothetical fragment lengths of the computer analyzed 16S rRNA sequences from the type strains of the ruminal spirochetes Treponema bryantii and T. saccharophilum confirmed the tentative novel identification. Transmission electron microscopy showed that the bacterium has the typical spirochetal structures, i.e. the outer sheath, the protoplasmic cylinder and the axial filament (it is not yet clear how many flagella compose the filament). An additional extracellular structure was observed which appeared as an exocytoplasmic polar flagellum, approximately 2 microns long and protruding from one tip of the cell. The average size of the cells was 0.5 x 10-15 microns and the wavelengths and the amplitudes of the primary coils were 2.9 and 1.3 microns, respectively.
Abstract: Crustaceans form a variety of calcium deposits in which they store calcium necessary for the mineralization of their exoskeletons. Calcium bodies, organs containing large amounts of calcium, have been reported in some terrestrial isopod crustaceans, but have not yet been extensively studied. We analyzed the architecture of these organs during the molt cycle in the isopod Titanethes albus. Two pairs of calcium bodies are positioned ventrolaterally in posterior pereonites of T. albus. Individual organs are epithelial sacs that contain material arranged in concentric layers delimited by thin laminae. As demonstrated by electron microscopy and fluorescence in situ hybridization, abundant bacteria are present within the calcium bodies. Regardless of the molt cycle stage, crystalline concretions are present in the central areas of the calcium bodies. Energy dispersive X-ray spectrometry of the concretions demonstrated that they are composed predominantly of calcium and phosphorus and selected area electron diffraction indicated the presence of hydroxyapatite. In molting animals, a glassy layer of mineralized matrix is formed between the envelope and the outermost lamina of the calcium body. This layer consists of an amorphous calcium mineral which contains less phosphorus than the central concretions and is resorbed after molt. Since changes in the mineralized matrix are synchronized with the molt cycle, the calcium bodies likely function as a storage compartment that complements sternal deposits as a source of calcium for the mineralization of the exoskeleton. Bacteria associated with the mineralized matrix of calcium bodies are evidently involved in calcium dynamics. (C) 2012 Elsevier Inc. All rights reserved.
