Genetic characterization of Shigatoxigenic and enteropathogenic Escherichia coli O80:H2 from diarrhoeic and septicaemic calves and relatedness to human Shigatoxigenic E. coli O80:H2.

AIM: The purpose of this work was to identify and genetically characterize enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) O80:H2 from diarrhoeic and septicaemic calves in Belgium and to comparing them with human EHEC after whole genome sequencing. METHODS AND RESULTS: Ten EHEC and 21 EPEC O80 identified by PCR between 2009 and 2018 from faeces, intestinal content and a kidney of diarrhoeic or septicaemic calves were genome sequenced and compared to 19 human EHEC identified between 2008 and 2019. They all belonged to the O80:H2 serotype and ST301, harboured the eaeξ gene, and 23 of the 29 EHEC contained the stx2d gene. Phylogenetically, they were distributed in two major sub-lineages: one comprised a majority of bovine EPEC whereas the second one comprised a majority of stx2d bovine and human EHEC. CONCLUSIONS: Not only EPEC but also EHEC O80:H2 are present in diarrhoeic and septicaemic calves in Belgium and are genetically related to human EHEC. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings support the need to assess cattle as potential source of contamination of humans by EHEC O80:H2 and to understand the evolution of bovine and human EHEC and EPEC O80:H2.

Effect of spinal cord stimulation on regional myocardial perfusion assessed by positron emission tomography.

Spinal cord stimulation (SCS) can relieve symptoms in patients with severe angina pectoris refractory to conventional medical or surgical therapy. This symptomatic improvement may result from decreased myocardial ischemia. To test this hypothesis, positron emission tomography (PET) and potassium-38 as a flow tracer were used in 8 patients for the quantitative evaluation of regional myocardial perfusion at rest and after exercise, before and during SCS. Potassium uptake was evaluated as myocardial clearance (flow times net extraction) in ml/min/100 g. Tomographic segments were categorized as nonaffected and affected on the basis of the absence or presence of arterial stenosis on coronary angiography and on the basis of thallium scintigraphic data. In nonaffected segments, before SCS, regional myocardial clearance significantly increased from rest (28 +/- 4) to exercise (47 +/- 13 clearance units; p less than 0.004). A similar increase occurred after SCS. In affected segments, before SCS, regional myocardial clearance barely increased (p = 0.065) from rest (26 +/- 6) to exercise (33 less than or equal to 12). In comparison, after SCS, the resting regional myocardial clearance was slightly elevated (29 +/- 8) reflecting an increased double product, but did not increase (p = 0.192) with exercise (34 +/- 12). However, the magnitude and duration of ST-segment depression decreased during treatment with SCS. Anginal pain occurred in all patients during control exercise, but was attenuated in all but one with SCS. These results indicate that SCS improves exercise-induced angina and electrocardiographic signs of ischemia but this influence does not appear to be mediated by changes in regional myocardial perfusion.

Dynamic graciloplasty for treatment of faecal incontinence.

Serious faecal incontinence due to anal sphincter damage should be treated by surgery. Graciloplasty has had limited success because the gracilis is a fast-twitch muscle and fatigues quickly. A favourable outcome in a patient who had dynamic (electrically stimulated) graciloplasty encouraged us to further assess this procedure. Gracilis muscle transposition was done in ten patients with complete anal incontinence due to anal atresia, sphincter damage, or neurogenic causes, and who had had several other unsuccessful treatments. 6 weeks after muscle transposition, intramuscular leads were implanted and connected to an implantable electric stimulator. Eight patients became continent, one patient still has a diverting colostomy, and a fistula developed in the other patient. Anal sphincter pressure improved from 35 mm Hg without stimulation to 62 mm Hg with stimulation at 8 weeks (mean increase 28 mm Hg [95% confidence interval 18, 36], p less than 0.01). Retention time of a phosphate enema increased from 22 to 281 s (mean increase 259 s [82, 436], p less than 0.01). Defaecography showed that the new sphincter was functioning. Defaecation was possible when the stimulator was turned "off" with a magnet. Dynamic graciloplasty can restore continence and it improves quality of life in faecally incontinent patients for whom other treatments have been unsuccessful.

Abnormalities in the spontaneous firing patterns of cultured rat neocortical neurons after chronic exposure to picrotoxin during development in vitro.

We have used the GABA-A antagonist picrotoxin (PTX) to investigate whether chronic disinhibition, leading to intensified neuronal firing, would induce a specific pattern of physiological alterations in cultured rat neocortex cells. Overall mean spontaneous discharge rates were little affected by 1 microM PTX but firing occurred mainly as repetitive high-frequency bursts of action potentials. This "phasic" pattern contrasted with the irregular, quasi-random, firing usually seen in control units. Neurons tested in normal growth medium after prolonged exposure to 1 microM PTX showed weaker interspike interval dependencies (Markov value) than in controls, along with reduced regularity in the occurrence of bursts. Since all physiological changes were opposite in direction to those reported earlier after chronic suppression of bioelectric activity, the results support the hypothesis that endogenous synaptic and/or action potentials are important for the maturation of neocortical networks. Since experimental alterations were found only in spike-train parameters which reflect ontogenetic changes in untreated control cultures, GABAergic inhibition (by preventing neuronal discharges from becoming too intense) presumably serves to constrain the rate of development within optimal limits.

Development in the absence of spontaneous bioelectric activity results in increased stereotyped burst firing in cultures of dissociated cerebral cortex.

Quantitative analysis of neuronal firing patterns was used to study the effects of chronic suppression of bioelectric activity (BEA) on functional development in primary cultures of fetal rat cerebral cortex. BEA was monitored with extracellular electrodes in active control cultures or, after return to control medium, in cultures chronically silenced with tetrodotoxin (TTX) at around 7, 14, 21 and 42 days in vitro. Spike trains of single neurons lasting up to 25 min duration were analyzed using a previously published set of computer programs. In control cultures, the main developmental trends seen in a previous study could be replicated. After development in the presence of TTX, activity levels were increased at all ages, and a high incidence was found of a single firing pattern characterized by stereotyped burst firing, while showing a low minute order variability in firing rate and low dependencies between successive intervals; conversely, the incidence of variable/non-burst firing was decreased relative to untreated cultures. The former firing pattern (i.e. non-variable bursting) could also be produced through acute addition of the GABA (A)-antagonist picrotoxin to control cultures, and resembled interictal burst firing observed in models of chronic epilepsy in vivo. These similarities suggest that chronic silencing of the cultures may have resulted in a functional disinhibition of the neuronal network; such disinhibition might be related to the increased cell death which we observed with chronic TTX-treatment in the same cultures.

Accelerated neural network formation in rat cerebral cortex cultures chronically disinhibited with picrotoxin.

Our aim was to determine if chronic blockade of GABAergic inhibitory synaptic activity, monitored electrophysiologically at the neuronal level, would affect synapse formation and ultrastructure in dissociated fetal rat cerebral cortex cultures. This was achieved by adding picrotoxin to the serum-free growth medium in a dose that induced continuous epileptiform discharges throughout the culture period. Light and electron microscopic analysis suggested an accelerated synaptic network formation in the experimental cultures during the first 2 weeks in vitro. The elimination of excess synapses (mainly on spines), which normally takes place during the fourth week in vitro, occurred 1 week earlier in the presence of picrotoxin. Finally, the experimental cultures showed smaller spine synapses throughout the entire culture period. Because these effects were opposite those induced by chronic tetrodotoxin-blockade of spontaneous bioelectric activity in a previous study, the underlying causal factor could be the respective intensification and suppression of neuronal activity in the two experiments. An appropriate balance between excitatory and inhibitory synaptic drive seems therefore to be important for normal maturation of neocortical circuitry.

Spontaneous neuronal firing patterns in fetal rat cortical networks during development in vitro: a quantitative analysis.

The development of spontaneous bioelectric activity (SBA) was studied in dissociated occipital cortex cultures prepared from 19 day old rat fetuses. All cultures, recorded one per diem from 5 to 33 days in vitro (div), showed SBA. Computer analysis of 76 extracellularly recorded single unit spike trains was carried out after selection on the basis of stationarity criteria. Statistically significant developmental trends were found in (i) interspike interval dependencies and (ii) fluctuations in mean firing rate, on the order of a minute or longer. The highly dependent firing patterns, including stereotyped bursting, were present mostly in the 9-12 div group, whereas minute-to-minute fluctuations in the intensity of firing were considerably more pronounced in the oldest group (22-33 div) than in the younger cultures. In addition, firing categories defined on the basis of factor-analysis revealed that such fluctuations were almost exclusively to be found in neurons which fired in a pronounced 'burst', rather than a relatively continuous fashion. Only a few mature appearing synaptic structures were observed electron microscopically prior to 12 div, but increased steadily in number thereafter. No cultures prior to 14 div, but all cultures older than this, stained positively for the presence of glutamic acid decarboxylase. An extensive immunoreactive, putative GABAergic, network was present by three weeks in vitro.

Bioelectric activity is required for regional specificity of sensory ganglion projections to spinal cord explants cultured in vitro.

Chronic blockade of spontaneous nerve impulses by means of tetrodotoxin leads to abnormally diffuse afferent projections into spinal cord cross-sections cultured for two to six weeks in vitro. In addition, even untreated explants which show a low level of spontaneous cord discharges failed to develop the normal degree of dorsal pathway selectivity. It is therefore concluded that centrally generated neuronal activity may play an important role in eliminating 'exuberant' connections which, during early development, are transiently present in this part of the nervous system.

Regional specificity of functional sensory connections in developing spinal cord cultures varies with the incidence of spontaneous bioelectric activity.

Cultured spinal cord explants in which little spontaneous bioelectric activity was present showed, when monitored using sensory ganglion-evoked monosynaptic action potentials, diffuse innervation by ingrowing afferent fibers at 3-4 weeks in vitro. In contrast, highly active cultures of the same age showed a strong tendency for functional sensory connections to be made within the dorsal half of the cord. Regional specificity was present in mature cultures (4-5 weeks in vitro), however, even when their spontaneous activity level was low. The results support earlier results using tetrodotoxin, and make it appear likely that centrally generated neuronal discharges can influence the topography of afferent terminals within the developing spinal cord.

Synaptogenesis in rat cerebral cortex cultures is affected during chronic blockade of spontaneous bioelectric activity by tetrodotoxin.

Reaggregated occipital cortex cells of 19-day-old fetal rats were grown in a serum-free, chemically defined medium, and chronically exposed to impulse-blocking levels of tetrodotoxin (TTX) in order to study the role of bioelectric activity in synaptogenesis. As judged by phase-contrast microscopy, no differences were noticed in the development of neuronal networks in the TTX-treated vs control cultures. In addition, when TTX was withdrawn from experimental cultures at any stage of development, bioelectric activity qualitatively comparable to that of the control cultures appeared within 1 min. However, quantitative stereological EM analysis revealed a significant retardation in synapse formation and ultrastructural maturation of synaptic junctions during the first 3 weeks. Around 23 days in vitro, the central zone of the reaggregates in control cultures started to degenerate, but not earlier then day 27 in TTX-treated cultures. During this time, the control, but not the experimental cultures showed (in intact tissue regions mainly situated at the outside of the aggregates) a large and selective loss of spine synapses. It is concluded that functional blockade not only retards the early growth and maturation of synaptic networks but also prevents the later occurring selective loss of spine synapses.

Effects of chronic suppression of bioelectric activity on the development of sensory ganglion evoked responses in spinal cord explants.

Dorsal root ganglion (DRG) afferent terminals were identified, using electrophysiological techniques, within fetal mouse spinal cord cross-sections cultured in vitro. Afferent distribution patterns were monitored in explants grown for 3 to 6 weeks either in a serum-supplemented or in a serum-free, chemically defined medium (CDM). Bioelectrically active control explants from both series were compared with explants which had been reversibly silenced by chronic exposure to tetrodotoxin (TTX). The control (serum-grown) cultures showed a significant dorsal cord innervation preference, whereas in the corresponding TTX series there was an equal dorsoventral distribution. In the CDM series the mean number of DRG evoked responses was lower at first in TTX-grown than in control cultures, but with age in vitro there was a rise in excitability to normal levels. Spontaneous neuronal activity was abnormally low in cultures (serum as well as CDM-grown) which had been exposed to TTX. It is concluded that bioelectric activity may be an important factor in the proper regulation of synaptic connectivity and functional responsiveness in the developing spinal cord.

Influence of growth medium, age in vitro and spontaneous bioelectric activity on the distribution of sensory ganglion-evoked activity in spinal cord explants.

The role of serum added to the culture medium and of spontaneous bioelectric activity in the development of sensory afferent connections was studied, employing fetal mouse spinal cord explants with attached dorsal root ganglia (DRG) as an in vitro model system. Afferent DRG terminals in the cord explants were localized on the basis of 'fixed-latency' DRG-evoked action potentials, which were anatomically verified in several experiments using horseradish peroxidase histology. In serum-supplemented medium (HSSM), but not in chemically defined medium (CDM), those DRG fibers which grew into the dorsal side of the cord terminated predominantly within the dorsal cord region, and remained there throughout the experimental period (18-33 days in vitro). In contrast, ventrally entering fibers terminated equally in both the dorsal and the ventral cord regions in young cultures (18-24 days in vitro) but were no longer observed after 27 days in vitro. Cultures grown in HSSM with the addition of xylocaine, in order to chronically suppress spontaneous bioelectric activity, essentially corresponded (at 25-32 days in vitro) to the picture seen in the control series at the same age. On the basis of polysynaptic DRG-evoked responses in the cord, developmental changes in local neuronal networks were inferred which resulted in less spread of DRG-evoked activity with age in HSSM, and more spread with age in CDM-grown cultures. It is concluded that for the formation of selective DRG connections in the spinal cord: (i) a serum-borne factor plays a role: and (ii) functional activity is not required.

Nerve outgrowth, synaptogenesis and bioelectric activity in fetal rat cerebral cortex tissue cultured in serum-free, chemically defined medium.

Dissociated and non-dissociated cerebral cortex of fetal rat was successfully cultured in a serum-free, chemically defined medium for at least 18 days without any preincubation in serum-supplemented medium. Neurite outgrowth, synapse formation and spontaneous bioelectric activity, in its qualitative and quantitative aspects, were essentially the same as was observed in medium containing 20% heat-inactivated horse serum.

A quantitative electron microscopic study on synapse formation in dissociated fetal rat cerebral cortex in vitro.

Occipital cortex of 19-day-old fetal rats was dissociated and cultured in vitro for 2-5 weeks in horse serum supplemented Eagle's MEM. The outgrowing neurons rapidly formed a dense network which started to degenerate after 3 weeks in vitro. By means of electron microscopy the numerical development of 6 different categories of synapses was followed during the time in culture. This approach revealed a sigmoid growth curve emerging over the first 3 weeks for the category of axo-dendritic synapses (which comprised the bulk of all synapses counted). Thereafter a decline in number set in. Chronic exposure of these dissociated cerebral cortex cultures to 50 microgram/ml xylocaine (a concentration adequate to block all measurable bioelectrical activity) did not prevent the formation of functional synaptic networks having normal synaptic ultrastructure. These results are in agreement with previous studies on fetal cerebral explants in culture. However, in some groups of our cultures, xylocaine led to a retardation in neurite outgrowth and in numerical synapse formation. Since these xylocaine effects were dose-related at a concentration double that required to silence the cultures, the growth retardation was probably not due to selective suppression of bioelectric activity, but rather to some general cytotoxic effect of the drug. In one of the xylocaine-treated groups (50 microgram/ml) which showed an exceptionally rapid neuronal outgrowth by the use of horse serum obtained from a different source than in the other groups, no deficit was noted in the number of synapses formed. Extracellular recording in this latter drug-treated group of cultures during the third week revealed (after return to control medium) spontaneous isolated action potentials, burst patterns and slow waves which were indistinguishable from the bioelectric activity seen in the control cultures. It is concluded that bioelectric activity in dissociated cortex cultures is not a prerequisite for the formation of apparently normal, functional synaptic networks.

Chemically defined medium enhances bioelectric activity in mouse spinal cord-dorsal root ganglion cultures.

Co-cultures of mouse spinal cord with dorsal root ganglion (DRG) cultures were grown either in horse serum (HS)-supplemented medium or in a serum-free, chemically defined medium (CDM). The cytoarchitecture of cord--DRG explants was fully retained in CDM, with little or no distortion due to flattening of the explant, as is invariably observed in HS-supplemented cultures. Functional properties such as bioelectric activity and DRG--spinal cord interconnectivity were well sustained in CDM.

Autoradiography of the olfactory-hippocampal pathway in the cat with special reference to the perforant path.

The anatomical pathway from the prepyriform cortex to the hippocampus in the cat was traced autoradiographically by means of anterograde transport of [3H] leucine. A direct projection from the prepyriform cortex to the lateral entorhinal area was confirmed in the cat: the termination of these fibers was largely confined to the outer part of the molecular layer. From the lateral entorhinal area, the perforant path fibres terminate on the most distal parts of the dendrites of the hippocampal granule and pyramidal cells. However, differences between cat and rat were found with respect to the terminations in the CA1 area.

An olfactory input to the hippocampus of the cat: field potential analysis.

Hippocampal responses to electrical stimulation of the prepyriform cortex in the cat were studied both in acute experiments under halothane anesthesia and in awake cats with chronically indwelling electrodes. Analysis of field potentials and unit activity indicated the extent to which different hippocampal subareas were activated, the laminar level at which the synaptic action took place and the dynamics of the evoked responses. It was found that: (1) the main generator of evoked responses in the hippocampus upon prepyriform cortex stimulation is localized in the fascia dentata and CA3 (CA1 pyramidal cells, and probably also subiculum cells, are activated but in a lesser degree); (2) the initial synaptic activity takes place at the most distal part of the dendrites of fascia dentata granuhat leads to a transient increase in the firing rate of the hippocampal units, which is often followed by a long-lasting decrease in firing rate. We conclude that the pathway from the prepyriform cortex via lateral entorhinal cortex to hippocampal neurons may enable olfactory inputs to effectively excite hippocampal neurons.