Co-circulating microorganisms in questing Ixodes scapularis nymphs in Maryland

Ixodes scapularis can be infected with Borrelia burgdorferi, Anaplasma phagocytophilum, Bartonella spp., Babesia microti, and Rickettsia spp., including spotted-fever group Rickettsia. As all of these microorganisms have been reported in Maryland, the potential for these ticks to have concurrent infections exists in this region. To assess the frequency of these complex infections, 348 I. scapularis nymphs collected in 2003 were screened for these microorganisms by PCR with positives being confirmed by DNA sequencing. Borrelia burgdorferi was detected in 14.7% of nymphs. Anaplasma phagocytophilum (0.3%), Rickettsia spp. (19.5%), and an uncategorized agent (0.9%) was also detected. Dual infections were detected with B. burgdorferi and Rickettsia spp. as well as a triple infection with B. burgdorferi, Rickettsia spp., and an uncategorized agent. Infections with B. burgdorferi and Rickettsia spp. were statistically independent of one another. However, infection with B. burgdorferi and any one of these other microorganisms appears to occur more frequently than by chance alone, probably as a result of shared enzootic cycles. This study confirms that multiple microorganisms co-circulate with B. burgdorferi in I. scapularis in Maryland and demonstrates that Rickettsia spp. and B. burgdorferi circulate independently and at nearly equal frequencies, while A. phagocytophilum and other unrecognized organisms are less common.     

Bacteriophage resistance of attached organisms as a factor in the survival of plasmid-bearing strains of Escherichia coli

Unattached organisms of plasmid-free and plasmid-bearing strains of Escherichia coli showed marked sensitivity to phages T4, Tula and K3 on incubation in broth at 37°C but organisms attached to glass beads or sand were resistant. Phage T4 sensitivity of free organisms and resistance of glass bead-attached ones were also observed when incubation was in broth at 15° or 20°C or anaerobically at 37°C. In contrast, free and attached organisms were resistant at 37°C or lower temperature when incubation was in poor media. It seems likely that the presence of phage will be a major factor reducing survival in the intestine and in sewage and that attachment (which is more significant for strains bearing certain plasmids) will protect. In contrast, survival of either free or attached organisms in polluted water will probably not be significantly influenced by the presence of phage.     

Analysis of Major Surface Polypeptides of Rickettsia japonica

Major surface polypeptides of Rickettsia japonica migrated to the position of 120, 135, and 145 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, when the organisms were solubilized at room temperature. Two major bands at the position of 135 and 185 kDa were seen, when the organisms were solubilized by heating before electrophoresis. Heat-denaturation of the 120- and 145-kDa polypeptides in excised gel bands changed their mobility and caused them to migrate to 135- and 185-kDa positions, respectively. Two polypeptides at the 120-kDa position were demonstrated: one is a major heat-modifiable polypeptide and the other a minor heat-stable. Peptide mapping was performed to determine the identity between native and denatured polypeptides.     

Protein biosynthesis changes in Trypanosoma cruzi induced by supra-optimal temperature

Early during vertebrate infection, T. cruzi is exposed to the host blood at an elevated temperature. Bearing this in mind, the pattern of protein synthesis of two parasite forms was examined. SDS-PAGE of heated organisms showed an increase in at least four proteins (103, 92, 75 and 61 kD). The temperature effect is also manifested in cells whose RNA synthesis is reduced by actinomycin D treatment. The synthesis of the '29 degrees proteins' is inhibited at 40 degrees C in organisms growing in culture medium; when the organisms were maintained in serum, the inhibition was not observed. The inhibitory effect observed at 40 degrees C was reversed when the temperature was shifted to 29 degrees C. These proteins were synthesized for 180 min at 37 degrees C or 360 min at 40 degrees C. The increased protein synthesis manifested at 37 degrees C had decreased 45 min after the temperature was lowered to 29 degrees C. When the cells were pre-incubated at 40 degrees C and shifted to 29 degrees C, the synthesis of the heat-induced proteins proceeded for at least 180 min. This pattern of heat induction in epimastigotes and trypomastigotes is the same irrespective of whether the incubation medium is LIT (for epimastigotes), M-16 (for trypomastigotes), or when serum was used for both cell types.     

Diversity and infection prevalence of endosymbionts in natural populations of the chestnut weevil: relevance of local climate and host plants

Many insects are ubiquitously associated with multiple endosymbionts, whose infection patterns often exhibit spatial and temporal variations. How such endosymbiont variations are relevant to local adaptation of the host organisms is of ecological interest. Here, we report a comprehensive survey of endosymbionts in natural populations of the chestnut weevil Curculio sikkimensis, whose larvae are notorious pests of cultivated chestnuts and also infest acorns of various wild oaks. From 968 insects representing 55 localities across the Japanese Archipelago and originating from 10 host plant species, we identified six distinct endosymbiont lineages, namely Curculioniphilus, Sodalis, Serratia, Wolbachia, Rickettsia and Spiroplasma, at different infection frequencies (96.7%, 12.8%, 82.3%, 82.5%, 28.2% and 6.8%, respectively) and with different geographical distribution patterns. Multiple endosymbiont infections were very common; 3.18±0.61 (ranging from 1.74 to 5.50) endosymbionts per insect on average in each of the local populations. Five pairs of endosymbionts (Curculioniphilus-Serratia, Curculioniphilus-Wolbachia, Sodalis-Rickettsia, Wolbachia-Rickettsia and Rickettsia-Spiroplasma) co-infected the same host individuals more frequently than expected, while infections with Serratia and Wolbachia were negatively correlated to each other. Infection frequencies of the endosymbionts were significantly correlated with climatic and ecological factors: for example, higher Sodalis, Wolbachia and Rickettsia infections at localities of higher temperature; lower Wolbachia and Rickettsia infections at localities of greater snowfall; and higher Curculioniphilus, Sodalis, Serratia, Wolbachia and Rickettsia infections on acorns than on chestnuts. These patterns are discussed in relation to potential host-endosymbiont co-evolution via local adaptation across geographical populations.     

Temperature-dependent changes in fluid transport across goldfish gallbladder

Summary The temperature dependence of fluid transport acrossin vitro preparations of goldfish gallbladder was measured using a gravimetric technique. Fluid transport showed a direct dependence on incubation temperature when the adaptation temperature was kept constant. For constant incubation temperature, transport fell as the adaptation temperature rose. The width of intercellular channels varied with incubation and adaptation temperature as expected if fluid were to cross the tissue by this route. The structure of the gallbladder was otherwise unaffected by changes of temperature. Intracellular concentrations of Na, K and Cl also depended on the environmental temperature of the fish. The levels of Na and Cl increased and the level of K decreased, at constant incubation temperature, as the adaptation temperature rose from 8 to 30°C. These changes took two to three weeks to become apparent while fluid transport regulated within 20 hours of raising the environmental temperature. The osmotic permeability of the gallbladder remained independent of both incubation and adaptation temperature.The outcome of adaptation was to maintain constant both the ionic composition of the epithelium and the rate at which it could transport fluid, when these parameters were measured at incubation temperatures equal to the previous environmental temperature of the fish. The significance of these findings is discussed and a mechanism for regulation postulated which involves an initial regulation of salt entry into the mucosa followed by long term changes in the pumping ability of newly synthesized cells.     

Transmission electron microscopy as a tool for exploring bacterial proteins: model of RickA in Rickettsia conorii

Rickettsia conorii, the etiologic agent of Mediterranean spotted fever, belongs to the spotted fever group of Rickettsia. It is an obligate intracellular bacterium that grows within the cytoplasm of its eukaryotic host cells. It is motile in the cytoplasm of infected cells and RickA is reported as critical protein in this aspect. However, the subcellular localization of RickA remains uncertain. We describe a simple method allowing RickA protein to be localized by immunofluorescence assay (IFA) and transmission electron microscopy (TEM). By using IFA we showed the global expression of surface protein RickA in R. conorii organisms. The TEM results showed that RickA is widely expressed over the entire bacterial surface of R. conorii.     

'Sloughing-off' of heterochromatin in Werner's syndrome cells during high-temperature phosphate incubation

Previous investigations of cells undergoing rapid division revealed the presence of heterochromatic 'dots' in chromosomes as well as numerous chromocentres in interphase nuclei. Such structures were seen in human embryonic cells, as well as cells from organisms capable of regeneration, and cells from various malignancies. Cells with a reduced capacity for reproduction were found to be virtually devoid of nuclear chromocentres and chromosome dots after incubation in phosphate buffer at high temperature. The lack of heterochromatin in such cells (Werner's syndrome) thereby explained their reduced capacity for cell division and the resultant rapid rate of aging in individuals afflicted. Re-examination of such slides containing these cells revealed that chromocentres and chromosome dots were present initially, but the incubation process resulted in a 'sloughing-off' of such structures. The incubation process left these heterochromatic structures intact in malignant and control cells, inferring a link between cell proliferation and stable intact heterochromatin. These findings implicate heterochromatin as the object of the purported chromosomal instability factor characteristic of Werner's syndrome. The loss of heterochromatin did not result in chromosome breakage, suggesting that heterochromatin may not be an integral part of chromosome structure, but rather a surface feature or covering.     

Rickettsia symbiont in the pea aphid Acyrthosiphon pisum: novel cellular tropism, effect on host fitness, and interaction with the essential symbiont Buchnera

In natural populations of the pea aphid Acyrthosiphon pisum, a facultative bacterial symbiont of the genus Rickettsia has been detected at considerable infection frequencies worldwide. We investigated the effects of the Rickettsia symbiont on the host aphid and also on the coexisting essential symbiont Buchnera. In situ hybridization revealed that the Rickettsia symbiont was specifically localized in two types of host cells specialized for endosymbiosis: secondary mycetocytes and sheath cells. Electron microscopy identified bacterial rods, about 2 mum long and 0.5 mum thick, in sheath cells of Rickettsia-infected aphids. Virus-like particles were sometimes observed in association with the bacterial cells. By an antibiotic treatment, we generated Rickettsia-infected and Rickettsia-eliminated aphid strains with an identical genetic background. Comparison of these strains revealed that Rickettsia infection negatively affected some components of the host fitness. Quantitative PCR analysis of the bacterial population dynamics identified a remarkable interaction between the coexisting symbionts: Buchnera population was significantly suppressed in the presence of Rickettsia, particularly at the young adult stage, when the aphid most actively reproduces. On the basis of these results, we discussed the possible mechanisms that enable the prevalence of Rickettsia infection in natural host populations in spite of the negative fitness effects observed in the laboratory.     

Bacteriological analysis of water by potentiometric measurement of lipoic acid reduction: preliminary assays for selective detection of indicator organisms

The practical task of adapting an original potentiometric technique to the bacteriological analysis of water is discussed. Various laboratory strains of organisms belonging to the usual aquatic flora were inoculated one by one in a minimal lactose broth supplied with lipoic (thioctic) acid. The time evolution of the redox potential of the cultures was followed during incubation by combined gold versus reference electrodes. When the incubation temperature was regulated at 36 degrees C, most organisms were able to grow and to reduce the coenzyme, generating changes in the redox potential of the culture. However, very few organisms developed significant reductive activity when the temperature was increased to 41 degrees C and when the broth was provided with sodium deoxycholate. Among the fecal coliform organisms, only Escherichia coli and Klebsiella pneumoniae exhibited early but reproducible potential-time responses. Positive potentiometric responses were also recorded with Acinetobacter calcoaceticus. E. coli showed rapid potentiometric signals as compared with K. pneumoniae. The time required for 100-mV shift of potential to be detected was related to the logarithm of the initial concentration of E. coli or K. pneumoniae in the culture broth. Experiments on natural surface water samples showed the the potentiometric method, associated with the selective incubation conditions, mainly detected E. coli among the bacterial flora of the tested environmental water. The calibration curve relating the time required for a 100-mV shift of potential to be detected to the number of fecal coliforms, as determined by control fecal coliform-selective plate counts, was consistent with the composite standard curve of detection times obtained with six different laboratory strains of E. coli.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bacteriological analysis of water by potentiometric measurement of lipoic acid reduction: preliminary assays for selective detection of indicator organisms.

The practical task of adapting an original potentiometric technique to the bacteriological analysis of water is discussed. Various laboratory strains of organisms belonging to the usual aquatic flora were inoculated one by one in a minimal lactose broth supplied with lipoic (thioctic) acid. The time evolution of the redox potential of the cultures was followed during incubation by combined gold versus reference electrodes. When the incubation temperature was regulated at 36 degrees C, most organisms were able to grow and to reduce the coenzyme, generating changes in the redox potential of the culture. However, very few organisms developed significant reductive activity when the temperature was increased to 41 degrees C and when the broth was provided with sodium deoxycholate. Among the fecal coliform organisms, only Escherichia coli and Klebsiella pneumoniae exhibited early but reproducible potential-time responses. Positive potentiometric responses were also recorded with Acinetobacter calcoaceticus. E. coli showed rapid potentiometric signals as compared with K. pneumoniae. The time required for 100-mV shift of potential to be detected was related to the logarithm of the initial concentration of E. coli or K. pneumoniae in the culture broth. Experiments on natural surface water samples showed the the potentiometric method, associated with the selective incubation conditions, mainly detected E. coli among the bacterial flora of the tested environmental water. The calibration curve relating the time required for a 100-mV shift of potential to be detected to the number of fecal coliforms, as determined by control fecal coliform-selective plate counts, was consistent with the composite standard curve of detection times obtained with six different laboratory strains of E. coli.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron Microscopy of the Cell Wall of Rickettsia prowazeki

Purified Rickettsia prowazeki were found to undergo morphological changes resembling plasmolysis when stained with uranyl acetate, resulting in rod-like forms. Sequential electron micrographs of disintegrating organisms provide evidence for the cell wall origin of these rod-like forms. The substructure of the cell wall was discerned by using negative-contrast electron microscopy. The wall was found to be composed of repetitive subunits with a periodicity of 13 nm and was surrounded by a thin membrane.     

Rapid homologous up-regulation of binding capacity of androgen receptors in intact cells

Incubation of MCF 7 cells with 5 alpha-dihydrotestosterone (DHT) at 37 degrees C led to a 70% increase in the Bmax of androgen receptor, as compared to the values measured at 2 degrees C, without detectable changes in equilibrium dissociation constants. When MCF 7 cells were incubated with hormone at 2 degrees C, to reach steady-state levels of androgen-receptor complex, a subsequent temperature shift to 37 degrees C induced a rapid (t 1/2 = 3 min) cycloheximide-insensitive increase in DHT binding to androgen receptor. MCF 7 cell treatments at 37 degrees C either before or after incubation with DHT at 2 degrees C showed that up-regulation of binding capacity of androgen receptor could be observed only if hormone is present during incubation at physiological temperature.     

Synchronous Activation of Cell Division by Light or Temperature Stimuli in the Dimorphic Yeast Schizosaccharomyces japonicus

Many fungi respond to light and regulate fungal development and behavior. A blue light-activated complex has been identified in Neurospora crassa as the product of the wc-1 and wc-2 genes. Orthologs of WC-1 and WC-2 have hitherto been found only in filamentous fungi and not in yeast, with the exception of the basidiomycete pathogenic yeast Cryptococcus. Here, we report that the fission yeast Schizosaccharomyces japonicus responds to blue light depending on Wcs1 and Wcs2, orthologs of components of the WC complex. Surprisingly, those of ascomycete S. japonicus are more closely related to those of the basidiomycete. S. japonicus reversibly changes from yeast to hyphae in response to environmental stresses. After incubation at 30°C, a colony of yeast was formed, and then hyphal cells extended from the periphery of the colony. When light cycles were applied, distinct dark- and bright-colored hyphal cell stripes were formed because the growing hyphal cells had synchronously activated cytokinesis. In addition, temperature cycles of 30°C for 12 h and 35°C for 12 h or of 25°C for 12 h and 30°C for 12 h during incubation in the dark induced a response in the hyphal cells similar to that of light. The stripe formation of the temperature cycles was independent of the wcs genes. Both light and temperature, which are daily external cues, have the same effect on growing hyphal cells. A dual sensing mechanism of external cues allows organisms to adapt to daily changes of environmental alteration.     

Osmotic Behavior of Bacterial Protoplasts: Temperature Effects

Among protoplasts released from cells of Bacillus megaterium grown at 20, 30, or 37 C, osmotic swelling in NaCl solution at a given external osmotic pressure was greatest for protoplasts from cells grown at 20 C and least for protoplasts from cells grown at 37 C. Protoplasts from cells grown at lower temperaturs were also less stable to osmotic shock and lysed at higher external osmotic pressures than did protoplasts from cells grown at higher temperatures. But for cells grown at any one temperature, osmotic stabilization was itself temperature dependent so that the higher the ambient incubation temperature, the higher the osmotic pressure needed to prevent lysis of a given fraction of the input protoplast population. However, comparison of the osmotic stability of protoplasts from cells grown at different temperatures at various ambient incubation temperatures revealed that, except at 5 C where no differences were discerned, protoplasts from cells grown at lower temperatures still lysed at higher osmotic pressures than did those from cells grown at higher temperatures. The apparent internal osmolality (28 to 31 atm) did not vary significantly among whole cells from the three growth temperatures. Therefore, the observed differences in osmotic behavior could not be attributed to changes in internal osmotic pressure. Rather, it seemed likely that the differences were due to changes in membrane properties.     

Actin-Based Motility of Intracellular Microbial Pathogens

A diverse group of intracellular microorganisms, including Listeria monocytogenes, Shigella spp., Rickettsia spp., and vaccinia virus, utilize actin-based motility to move within and spread between mammalian host cells. These organisms have in common a pathogenic life cycle that involves a stage within the cytoplasm of mammalian host cells. Within the cytoplasm of host cells, these organisms activate components of the cellular actin assembly machinery to induce the formation of actin tails on the microbial surface. The assembly of these actin tails provides force that propels the organisms through the cell cytoplasm to the cell periphery or into adjacent cells. Each of these organisms utilizes preexisting mammalian pathways of actin rearrangement to induce its own actin-based motility. Particularly remarkable is that while all of these microbes use the same or overlapping pathways, each intercepts the pathway at a different step. In addition, the microbial molecules involved are each distinctly different from the others. Taken together, these observations suggest that each of these microbes separately and convergently evolved a mechanism to utilize the cellular actin assembly machinery. The current understanding of the molecular mechanisms of microbial actin-based motility is the subject of this review.     

An ontogenetic shift in the response of heart rates to temperature in the developing snapping turtle (Chelydra serpentina)

The affect of acute changes in temperature on heart rates was investigated for the first time in a developing reptile. Heart rates were determined early and late in incubation in snapping turtle (Chelydra serpentina) eggs. Late in incubation heart rates at any given temperature were lower than those observed early in incubation. The results of temperature switching experiments late in incubation were consistent with thermal acclimation.     

Outer membrane protein composition of Yersinia pestis at different growth stages and incubation temperatures.

The protein composition of the outer membrane of Yersinia pestis grown at 26 and at 37 degrees C was examined. The outer membrane was isolated by isopycnic sucrose density centrifugation, and its degree of purity was determined with known inner and outer membrane components. Using two-dimensional gel electrophoresis, we identified a large number of heat-modifiable proteins in the outer membrane of cells grown at either incubation temperature. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of heated preparations indicated five proteins in the outer membrane of 37 degrees C-grown cells not evident in 26 degrees C-grown cells. Differences in the protein composition of the outer membrane due to the stage of growth were evident at both 26 degrees C and 37 degrees C, although different changes were found at each temperature. When cell envelopes were examined for the presence of peptidoglycan-associated proteins, no differences were seen as a result of stage of growth. Envelopes from 26 degrees C-grown cells yielded two peptidoglycan-associated proteins, E and J. Cells grown at 37 degrees C, however, also contained an additional protein (F) which was not found in either the bound or free form 26 degrees C. The changes in outer membrane protein composition in response to incubation temperature may relate to known nutritional and antigenic changes which occur under the same conditions.     

Low Temperature-Induced Cytoplasmic Acidosis in Cultured Mung Bean (Vigna radiata [L.] Wilczek) Cells

Cold-induced changes in vivo in the cytoplasmic pH of suspension-cultured cells of mung bean (Vigna radiata [L.] Wilczek) were investigated by fluorescence-ratio imaging cryomicroscopy with special reference to the variations in the chilling sensitivity of cells during the growth cycle. Because of the preferential localization of the fluorophore in the cytoplasm under specified conditions and the ideal response of fluorescence to pH, fluorescein diacetate allows measurements to be made of temporal changes in cytoplasmic pH at low temperature. A remarkable difference was demonstrated in the cold-induced changes in cytoplasmic pH between cells at the early and late stages of exponential growth. The cells at the early stage of exponential growth were most sensitive to chilling, and the cytoplasmic pH decreased dramatically within a short period of incubation at 0[deg]C, decreasing from 7.4 to 6.8 after 4 h and to 6.3 after 18 h. The cells at the late stage of exponential growth were chilling tolerant, and no significant decrease in the cytoplasmic pH was observed during the incubation at 0[deg]C for 24 h or even longer. From the results presented here, it appears that cold-induced cytoplasmic acidosis is characteristic of chilling-sensitive mung bean suspension-cultured cells.     

Rickettsia felis from cat fleas: isolation and culture in a tick-derived cell line

Rickettsia felis, the etiologic agent of spotted fever, is maintained in cat fleas by vertical transmission and resembles other tick-borne spotted fever group rickettsiae. In the present study, we utilized an Ixodes scapularis-derived tick cell line, ISE6, to achieve isolation and propagation of R. felis. A cytopathic effect of increased vacuolization was commonly observed in R. felis-infected cells, while lysis of host cells was not evident despite large numbers of rickettsiae. Electron microscopy identified rickettsia-like organisms in ISE6 cells, and sequence analyses of portions of the citrate synthase (gltA), 16S rRNA, Rickettsia genus-specific 17-kDa antigen, and spotted fever group-specific outer membrane protein A (ompA) genes and, notably, R. felis conjugative plasmids indicate that this cultivatable strain (LSU) was R. felis. Establishment of R. felis (LSU) in a tick-derived cell line provides an alternative and promising system for the expansion of studies investigating the interactions between R. felis and arthropod hosts.