Molecular identification of zoonotic hookworms in dogs from four counties of Kenya.

All canine hookworms are known to be zoonotic, causing infections ranging from transient skin irritations to prolonged 'creeping eruptions', eosinophilic enteritis and even patent intestinal infections. There is little information on canine hookworm species and their public health significance in sub-Saharan Africa. This study determined the prevalence and species of hookworms in dogs from different climatic zones of Kenya. Dog faecal samples were collected from the environment, and hookworm eggs were isolated by zinc chloride flotation and subjected to DNA extraction. Polymerase chain reaction (PCR) assays targeting the internal transcribed spacer (ITS) 1 and 2, 5.8S and 28S ribosomal RNA of Ancylostoma spp. and Uncinaria stenocephala were performed, and hookworm species were identified by PCR-restriction fragment length polymorphism (RFLP) or DNA sequencing. Hookworm eggs were detected by microscopy in 490/1621 (30.23%, 95% CI 28.01-32.54) faecal samples. Estimates of faecal prevalence were high in counties receiving higher rainfall (Narok 46.80%, Meru 44.88%) and low in those with a more arid climate (Isiolo 19.73%, Turkana 11.83%). In a subset of 70 faecal samples, Ancylostoma caninum (n = 59) was the most common species, followed by A. braziliense (n = 10) and A. cf. duodenale (n = 1). This study reports for the first time the detection of A. cf. duodenale in dog faeces and zoonotic hookworm species in Kenyan dogs. These findings emphasize the need for control measures such as enforcing laws for restraining stray dogs, regular deworming of dogs, and public health awareness programmes aimed at informing communities on outdoor use of footwear.

Unbalanced Peptidergic Inhibition in Superficial Neocortex Underlies Spike and Wave Seizure Activity.

Slow spike and wave discharges (0.5-4 Hz) are a feature of many epilepsies. They are linked to pathology of the thalamocortical axis and a thalamic mechanism has been elegantly described. Here we present evidence for a separate generator in local circuits of associational areas of neocortex manifest from a background, sleep-associated delta rhythm in rat. Loss of tonic neuromodulatory excitation, mediated by nicotinic acetylcholine or serotonin (5HT3A) receptors, of 5HT3-immunopositive interneurons caused an increase in amplitude and slowing of the delta rhythm until each period became the "wave" component of the spike and wave discharge. As with the normal delta rhythm, the wave of a spike and wave discharge originated in cortical layer 5. In contrast, the "spike" component of the spike and wave discharge originated from a relative failure of fast inhibition in layers 2/3-switching pyramidal cell action potential outputs from single, sparse spiking during delta rhythms to brief, intense burst spiking, phase-locked to the field spike. The mechanisms underlying this loss of superficial layer fast inhibition, and a concomitant increase in slow inhibition, appeared to be precipitated by a loss of neuropeptide Y (NPY)-mediated local circuit inhibition and a subsequent increase in vasoactive intestinal peptide (VIP)-mediated disinhibition. Blockade of NPY Y1 receptors was sufficient to generate spike and wave discharges, whereas blockade of VIP receptors almost completely abolished this form of epileptiform activity. These data suggest that aberrant, activity-dependent neuropeptide corelease can have catastrophic effects on neocortical dynamics.

Characterization of the Blastocystis-specific faecal IgA immune response in pigs.

Blastocystis is an intestinal protist found in many species including humans and pigs. It has a controversial pathogenesis and has been implicated as a potential cause of irritable bowel syndrome. Our previous studies identified pigs as potential animal models for blastocystosis by demonstrating that they were likely natural hosts of Blastocystis and can harbour subtypes (ST) in common with humans. Furthermore, our finding of a lack of intestinal histopathology associated with Blastocystis infection in pigs is also a consistent finding in examined infected humans. In this study, we aimed to identify and characterize the Blastocystis-specific mucosal IgA response in pigs by immunoblotting, using pig faecal antibodies and Blastocystis antigen. Faeces from 233 pigs representing three age groups (sows/boars, growers/weaners and piglets) and including five dexamethasone-immunosuppressed research pigs were tested. The majority (81·5%) of the pigs had faecal IgA reactivity against Blastocystis proteins of molecular weights of 17·5-120 kDa. Reactivity to a >250 kDa protein was found in 18·5% of pigs. Notably, immunosuppressed pigs and piglets were statistically more likely to have reactivity to this protein compared to growers/weaners and sows/boars, respectively. These results corroborate other findings suggesting that compromised immunity may predispose to blastocystosis and support our contention that pigs are potentially good models for pathogenesis studies.

Identification and treatment of Strongyloides stercoralis infection in a Boston Terrier dog from south-eastern Australia.

Strongyloides stercoralis, the causative agent of strongyloidiasis, is a potentially zoonotic intestinal nematode endemic to northern Australia. Strongyloidiasis is typically observed in immunocompromised hosts and is characterised by gastrointestinal signs, respiratory symptoms and a failure to thrive. In immunocompromised hosts, hyperinfection syndrome and disseminated infections can prove life-threatening. A 24-month-old Boston Terrier dog was referred for investigation of chronic small and large intestinal watery hematochezic diarrhoea, emaciation and hematemesis. Small intestinal histology identified a nematode despite consecutive negative faecal flotations. A real-time polymerase chain reaction and Baermann test subsequently confirmed infection with S. stercoralis. The dog had received an oral parasiticide comprising milbemycin oxime and afoxolaner every month for the 11 months prior to this diagnosis. Despite fenbendazole being reported as successful in the treatment of canine strongyloidiasis, a course of fenbendazole failed to clear the infection. Eradication of S. stercoralis infection was confirmed after the administration of off-label ivermectin fortnightly for 12 doses. Attention should be paid to this nematode as the failure of routine copromicroscopic methods to diagnose S. stercoralis infections can result in misdiagnosis, mistreatment and progression of the disease. Off-label ivermectin may be an alternative to fenbendazole for the treatment of Strongyloides spp. infection in dogs.

Blastocystis subtypes in symptomatic and asymptomatic family members and pets and response to therapy.

BACKGROUND: Blastocystis is a common, enteric parasite. The pathogenicity of the organism is uncertain, but subtypes (ST) 1 and 3 have been reported more likely to cause irritable bowel-like symptoms. AIMS: We treated symptomatic patients positive for Blastocystis with conventional therapy and analysed 16 small-subunit (SSU) rDNA to assess clearance and carriage rates and ST prevalence of the parasite in the asymptomatic household members. METHODS: In a longitudinal, prospective case study, 11 symptomatic patients positive for Blastocystis underwent outpatient clinical assessment to exclude other diagnoses before 14 days of either metronidazole 400 mg three times daily or trimethoprim/sulfamethoxazole 160/800 mg twice-daily therapy. Faecal specimens were collected from patients at baseline, day 15, 28 and 56 after therapy and from 17 family members and eight pets at day 15. Specimens were analysed using faecal smear, culture and polymerase chain reaction analysis of 16SSU rDNA. RESULTS: No patient cleared the organism following therapy. ST 1 (45%), 3 (36%), 4 (36%) and 6 (9%) were found in the symptomatic Blastocystis patients, and ST identified before and after therapy were identical in each individual. All household contacts were positive for Blastocystis and 16/17 (94%) contacts showed identical Blastocystis ST to the symptomatic family member. All pets were positive for Blastocystis with polymerase chain reaction testing, 7/8 (88%) demonstrating ST concordance with the symptomatic Blastocystis patients. CONCLUSIONS: Conventional therapy is ineffective for symptomatic Blastocystis infection. The high prevalence of Blastocystis infection within households suggested transmission between humans and their pets. Subtyping analysis of SSU rDNA alone in Blastocystis does not appear to predict pathogenicity.

Detection of Group 1 Trypanosoma brucei gambiense by loop-mediated isothermal amplification.

Trypanosoma brucei gambiense group 1 is the major causative agent of the Gambian human African trypanosomiasis (HAT). Accurate diagnosis of Gambian HAT is still challenged by lack of precise diagnostic methods, low and fluctuating parasitemia, and generally poor services in the areas of endemicity. In this study, we designed a rapid loop-mediated isothermal amplification (LAMP) test for T. b. gambiense based on the 3' end of the T. b. gambiense-specific glycoprotein (TgsGP) gene. The test is specific and amplifies DNA from T. b. gambiense isolates and clinical samples at 62°C within 40 min using a normal water bath. The analytical sensitivity of the TgsGP LAMP was equivalent to 10 trypanosomes/ml using purified DNA and ∼1 trypanosome/ml using supernatant prepared from boiled blood, while those of classical PCR tests ranged from 10 to 10(3) trypanosomes/ml. There was 100% agreement in the detection of the LAMP product by real-time gel electrophoresis and the DNA-intercalating dye SYBR green I. The LAMP amplicons were unequivocally confirmed through sequencing and analysis of melting curves. The assay was able to amplify parasite DNA from native cerebrospinal fluid (CSF) and double-centrifuged supernatant prepared from boiled buffy coat and bone marrow aspirate. The robustness, superior sensitivity, and ability to inspect results visually through color change indicate the potential of TgsGP LAMP as a future point-of-care test.

Cellular mechanism of neuronal synchronization in epilepsy.

Interictal spikes are a simple kind of epileptic neuronal activity. Field potentials and intracellular recordings observed during interictal spikes of penicillin-treated slices of the hippocampus were reproduced by a mathematical model of a network of 100 hippocampal neurons from the region including CA2 and CA3. The model shows that this form of neuronal synchronization arises because of mutual excitation between neurons, each of which is capable of intrinsic bursting in response to a brief input.

Model of the origin of rhythmic population oscillations in the hippocampal slice.

One goal of mammalian neurobiology is to understand the generation of neuronal activity in large networks. Conceptual schemes have been based on either the properties of single cells or of individual synapses. For instance, the intrinsic oscillatory properties of individual thalamic neurons are thought to underlie thalamic spindle rhythms. This issue has been pursued with a computer model of the CA3 region of the hippocampus that is based on known cellular and synaptic properties. Over a wide range of parameters, this model generates a rhythmic activity at a frequency faster than the firing of individual cells. During each rhythmic event, a few cells fire while most other cells receive synchronous synaptic inputs. This activity resembles the hippocampal theta rhythm as well as synchronized synaptic events observed in vitro. The amplitude and frequency of this emergent rhythmic activity depend on intrinsic cellular properties and the connectivity and strength of both excitatory and inhibitory synapses.

Transovarial transmission of murine typhus rickettsiae in Xenopsylla cheopis fleas.

It has been generally accepted that infected fleas do not pass on Rickettsia mooseri, or indeed any other known pathogen, to their progeny. It is reported here that such transovarial transmission does occur in laboratory-infected Xenopsylla cheopis fleas. By means of the direct fluorescent antibody test, Rickettsia mooseri was observed in cells of the hemolymph of infected fleas. As many as 11 percent of the adults and 2.9 percent of the larvae of the generation reared therefrom, had demonstrable rickettsiae. Moreover, batches of the F1 fleas were capable of transmitting the infection to more than 18 percent of the rats they infested. The data support the contention that Xenopsylla cheopis fleas play an important role in the maintenance of murine typhus in rats in nature.

Detecting seizure origin using basic, multiscale population dynamic measures: preliminary findings.

Many types of electrographic seizures are readily identifiable by direct visual examination of electroencephalographic or electrocorticographic recordings. This process can, however, be painstakingly slow, and much effort has been expended to automate the process using various dynamic properties of epileptiform waveforms. As methods have become more subtle and powerful they have been used for seizure subclassification, seizure prediction, and seizure onset identification and localization. Here we concentrate on the last, with reference to seizures of neocortical origin. We briefly review some of the methods used and introduce preliminary results from a very simple dynamic model based on key electrophysiological properties found in some seizure types: occurrence of very fast oscillations (sometimes called ripples), excess gamma frequency oscillations, electroencephalographic/electrocorticographic flattening, and changes in global synchrony. We show how this multiscale analysis may reveal features unique to seizure onset and speculate on the underlying cellular and network phenomena responsible.

Axo-axonal coupling. a novel mechanism for ultrafast neuronal communication.

We provide physiological, pharmacological, and structural evidence that axons of hippocampal principal cells are electrically coupled, with prepotentials or spikelets forming the physiological substrate of electrical coupling as observed in cell somata. Antidromic activation of neighboring axons induced somatic spikelet potentials in neurons of CA3, CA1, and dentate gyrus areas of rat hippocampal slices. Somatic invasion by these spikelets was dependent on the activation of fast Na(+) channels in the postjunctional neuron. Antidromically elicited spikelets were suppressed by gap junction blockers and low intracellular pH. Paired axo-somatic and somato-dendritic recordings revealed that the coupling potentials appeared in the axon before invading the soma and the dendrite. Using confocal laser scanning microscopy we found that putative axons of principal cells were dye coupled. Our data thus suggest that hippocampal neurons are coupled by axo-axonal junctions, providing a novel mechanism for very fast electrical communication.

Could plasticity of inhibition pattern pattern generators?

A modeling study shows that inhibitory synapse plasticity, guided by simple activity-dependent rules, can lead to appropriate phase relationships within an oscillating network.

Extracellular signal-regulated kinase activation in the spinal cord contributes to visceral hypersensitivity induced by craniofacial injury followed by stress.

BACKGROUND: We previously developed an animal model to examine mechanisms that underlie the emergence of visceral hypersensitivity modeling pain characteristics of temporomandibular disorder (TMD) patients with comorbid irritable bowel syndrome (IBS). In ovariectomized (OVx) rats with estradiol (E2) replacement, visceral hypersensitivity developed subsequent to masseter muscle inflammation followed by repeated forced swim (FS) stress. The purpose of this study was to investigate whether activation of extracellular signal-regulated kinase (ERK) in the spinal cord contributes to visceral hypersensitivity in this overlapping pain model. METHODS: In OVx with E2 replacement rats masseter muscle inflammation was followed by 3 day FS (comorbid condition). Depression-like behaviors were assessed by sucrose preference and in the elevated plus maze, and visceral sensitivity was measured by the visceromotor response (VMR) to colorectal distention. The protein level of ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2) in the L6-S2 dorsal spinal cord was analyzed by western blot. KEY RESULTS: FS stress decreased sucrose consumption in E2 replaced rats in sucrose preference test. The expression of p-ERK1/2 in the L6-S2 dorsal spinal cord increased significantly in E2 with comorbid rats. Intrathecal injection of mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor PD98059 blocked the visceral hypersensitivity induced by masseter muscle inflammation combined with FS stress. CONCLUSIONS & INFERENCES: These data indicate that ERK1/2 activation contributes to the visceral hypersensitivity evoked by craniofacial inflammation pain combined with stress. The results may provide a new therapeutic avenue for alleviating overlapping pain conditions.

Neuronal networks for induced '40 Hz' rhythms.

A fast, coherent EEG rhythm, called a gamma or a '40 Hz' rhythm, has been implicated both in higher brain functions, such as the 'binding' of features that are detected by sensory cortices into perceived objects, and in lower level processes, such as the phase coding of neuronal activity. Computer simulations of several parts of the brain suggest that gamma rhythms can be generated by pools of excitatory neurones, networks of inhibitory neurones, or networks of both excitatory and inhibitory neurones. The strongest experimental evidence for rhythm generators has been shown for: (1) neocortical and thalamic neurones that are intrinsic '40 Hz' oscillators, although synchrony still requires network mechanisms; and (2) hippocampal and neocortical networks of mutually inhibitory interneurones that generate collective 40 Hz rhythms when excited tonically.

Gamma-frequency oscillations: a neuronal population phenomenon, regulated by synaptic and intrinsic cellular processes, and inducing synaptic plasticity.

Neurons are extraordinarily complicated devices, in which physical and chemical processes are intercoupled, in spatially non-uniform manner, over distances of millimeters or more, and over time scales of < 1 msec up to the lifetime of the animal. The fact that neuronal populations generating most brain activities of interest are very large-perhaps many millions of cells-makes the task of analysis seem hopeless. Yet, during at least some population activities, neuronal networks oscillate synchronously. The emergence of such oscillations generates precise temporal relationship between neuronal inputs and outputs, thus rendering tractable the analysis of network function at a cellular level. We illustrate this idea with a review of recent data and a network model of synchronized gamma frequency (> 20 Hz) oscillations in vitro, and discuss how these and other oscillations may relate to recent data on back-propagating, action potentials, dendritic Ca2+ transients, long-term potentiation and GABAA receptor-mediated synaptic potentials.

The radiological properties of a novel lung tissue substitute.

Lung phantoms have been manufactured using commercially available, polyurethane foam products. Some of these materials are no longer available; therefore, a new lung tissue substitute was developed. The elemental composition and radiological properties of the new lung tissue substitute are described in this paper. Because the lung tissue substitute will be used to manufacture phantom lungs that will be used to evaluate chest counting systems, it is necessary to know the radiological properties of the material. These properties must be compared with reference materials and materials that have been used for lung phantoms in the past. The radiological properties of interest include the electron density, mean excitation energy, electron stopping power and photon mass attenuation coefficients. In all these properties, the calculated values for the new lung tissue substitute closely matched the calculated values of ICRU Publication 44 lung tissue. Good agreement was also found when the new lung tissue substitute was compared with the Griffith lung tissue substitute described by the ICRU. The new material was determined to be an excellent lung tissue substitute.

Quantitative detection of viable helminth ova from raw wastewater, human feces, and environmental soil samples using novel PMA-qPCR methods.

In this study, we have evaluated the efficacy of propidium monoazide quantitative polymerase chain reaction (PMA-qPCR) to differentiate between viable and non-viable Ancylostoma caninum ova. The newly developed method was validated using raw wastewater seeded with known numbers of A. caninum ova. Results of this study confirmed that PMA-qPCR has resulted in average of 88 % reduction (P < 0.05) in gene copy numbers for 50 % viable +50 % non-viable when compared with 100 % viable ova. A reduction of 100 % in gene copies was observed for 100 % non-viable ova when compared with 100 % viable ova. Similar reductions (79-80 %) in gene copies were observed for A. caninum ova-seeded raw wastewater samples (n = 18) collected from wastewater treatment plants (WWTPs) A and B. The newly developed PMA-qPCR method was applied to determine the viable ova of different helminths (A. caninum, A. duodenale, Necator americanus and Ascaris lumbricoides) in raw wastewater, human fecal and soil samples. None of the unseeded wastewater samples were positive for the above-mentioned helminths. N. americanus and A. lumbricoides ova were found in unseeded human fecal and soil samples. For the unseeded human fecal samples (1 g), an average gene copy concentration obtained from qPCR and PMA-qPCR was found to be similar (6.8 × 10(5) ± 6.4 × 10(5) and 6.3 × 10(5) ± 4.7 × 10(5)) indicating the presence of viable N. americanus ova. Among the 24 unseeded soil samples tested, only one was positive for A. lumbricoides. The mean gene copy concentration in the positively identified soil sample was 1.0 × 10(5) ± 1.5 × 10(4) (determined by qPCR) compared to 4.9 × 10(4) ± 3.7 × 10(3) (determined by PMA-qPCR). The newly developed PMA-qPCR methods were able to detect viable helminth ova from wastewater and soil samples and could be adapted for health risk assessment.

A model of high-frequency ripples in the hippocampus based on synaptic coupling plus axon-axon gap junctions between pyramidal neurons.

So-called 200 Hz ripples occur as transient EEG oscillations superimposed on physiological sharp waves in a number of limbic regions of the rat, either awake or anesthetized. In CA1, ripples have maximum amplitude in stratum pyramidale. Many pyramidal cells fail to fire during a ripple, or fire infrequently, superimposed on the sharp wave-associated depolarization, whereas interneurons can fire at high frequencies, possibly as fast as the ripple itself. Recently, we have predicted that networks of pyramidal cells, interconnected by axon-axon gap junctions and without interconnecting chemical synapses, can generate coherent population oscillations at >100 Hz. Here, we show that such networks, to which interneurons have been added along with chemical synaptic interactions between respective cell types, can generate population ripples superimposed on afferent input-induced intracellular depolarizations. During simulated ripples, interneurons fire at high rates, whereas pyramidal cells fire at lower rates. The model oscillation is generated by the electrically coupled pyramidal cell axons, which then phasically excite interneurons at ripple frequency. The oscillation occurs transiently because rippling can express itself only when axons and cells are sufficiently depolarized. Our model predicts the occurrence of spikelets (fast prepotentials) in some pyramidal cells during sharp waves.

Self-organized synaptic plasticity contributes to the shaping of gamma and beta oscillations in vitro.

gamma (30-70 Hz) followed by beta (10-30 Hz) oscillations are evoked in humans by sensory stimuli and may be involved in working memory. Phenomenologically similar gamma-->beta oscillations can be evoked in hippocampal slices by strong two-site tetanic stimulation. Weaker stimulation leads only to two-site synchronized gamma. In vitro oscillations have memory-like features: (1) EPSPs increase during gamma-->beta; (2) after a strong one-site stimulus, two-site stimulation produces desynchronized gamma; and (3) a single synchronized gamma-->beta epoch allows a subsequent weak stimulus to induce synchronized gamma-->beta. Features 2 and 3 last >50 min and so are unlikely to be caused by presynaptic effects. A previous model replicated the gamma-->beta transition when it was assumed that K(+) conductance(s) increases and there is an ad hoc increase in pyramidal EPSCs. Here, we have refined the model, so that both pyramidal-->pyramidal and pyramidal-->interneuron synapses are modifiable. This model, in a self-organized way, replicates the gamma-->beta transition, along with features 1 and 2 above. Feature 3 is replicated if learning rates, or the time course of K(+) current block, are graded with stimulus intensity. Synaptic plasticity allows simulated oscillations to synchronize between sites separated by axon conduction delays over 10 msec. Our data suggest that one function of gamma oscillations is to permit synaptic plasticity, which is then expressed in the form of beta oscillations. We propose that the period of gamma oscillations, approximately 25 msec, is "designed" to match the time course of [Ca(2+)](i) fluctuations in dendrites, thus facilitating learning.

Differential expression of synaptic and nonsynaptic mechanisms underlying stimulus-induced gamma oscillations in vitro.

Gamma frequency oscillations occur in hippocampus in vitro after brief tetani delivered to afferent pathways. Previous reports have characterized these oscillations as either (1) trains of GABA(A) inhibitory synaptic events mediated by depolarization of both pyramidal cells and interneurons at least in part mediated by metabotropic glutamate and acetylcholine receptors, or (2) field potential oscillations occurring in the near absence of an inhibitory synaptic oscillation when cells are driven by depolarizing GABA responses and local synchrony is produced by field effects. The aim of this study was to investigate factors involved in the differential expression of these synaptically and nonsynaptically gated oscillations. Field effects were undetectable in control recordings but manifested when slices were perfused with hypo-osmotic solutions or a reduced level of normal perfusate. These manipulations also reduced the amplitude of the train of inhibitory synaptic events associated with an oscillation and enhanced the depolarizing GABA component underlying the post-tetanic depolarization. The resulting field oscillation was still dependent, at least in part, on inhibitory synaptic transmission, but spatiotemporal aspects of the oscillation were severely disrupted. These changes were also accompanied by an increase in estimated [K(+)](o) compared with control. We suggest that nonsynaptic oscillations occur under conditions also associated with epileptiform activity and constitute a phenomenon that is distinct from synaptically gated oscillations. The latter remain a viable model for in vivo oscillations of cognitive relevance.