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.

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.

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.

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.

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.

Estradiol modulates visceral hyperalgesia by increasing thoracolumbar spinal GluN2B subunit activity in female rats.

BACKGROUND: We previously reported estrogen modulates spinal N-methyl-d-aspartate (NMDA) receptor processing of colorectal pain through changes in spinal GluN1 subunit phosphorylation/expression. The purpose of this study was to investigate whether spinal GluN2B containing NMDA receptors are involved in estrogen modulation of visceral pain processing. METHODS: Behavioral, molecular, and immunocytochemical techniques were used to determine spinal GluN2B expression/phosphorylation and function 48 h following subcutaneous injection of estradiol (E2) or vehicle (safflower oil, Saff oil) in ovariectomized rats in the absence or presence of colonic inflammation induced by mustard oil. KEY RESULTS: E2 increased the magnitude of the visceromotor response (VMR) to colorectal distention compared to Saff oil in non-inflamed rats. Intrathecal injection of the GluN2B subunit antagonist, Ro 25-6981, had no effect on the VMR in non-inflamed E2 or Saff oil rats. Colonic inflammation induced visceral hyperalgesia in E2, but not Saff oil rats. Visceral hyperalgesia in E2 rats was blocked by intrathecal GluN2B subunit selective antagonists. In inflamed rats, E2 increased GluN2B protein and gene expression in the thoracolumbar (TL), but not lumbosacral (LS), dorsal spinal cord. Immunocytochemical labeling showed a significant increase in GluN2B subunit in the superficial dorsal horn of E2 rats compared to Saff oil rats. CONCLUSIONS & INFERENCES: These data support the hypothesis that estrogen increases spinal processing of colonic inflammation-induced visceral hyperalgesia by increasing NMDA receptor activity. Specifically, an increase in the activity of GluN2B containing NMDA receptors in the TL spinal cord by estrogen underlies visceral hypersensitivity in the presence of colonic inflammation.

Canine tick-borne pathogens and associated risk factors in dogs presenting with and without clinical signs consistent with tick-borne diseases in northern Australia.

OBJECTIVES: To estimate the proportion of canine tick-borne disease (CTBD) pathogens in dogs from northern states of Australia presenting with and without clinical signs/laboratory abnormalities suggestive of CTBD and to evaluate associated risk factors. DESIGN: Client-owned dogs presented to a general practice clinic in the Northern Territory (NT; n = 138) and five referral hospitals in south-east Queensland (SEQ; n = 100) were grouped into CTBD-suspect and -control groups based on clinical and laboratory criteria. Blood and sera were screened for haemotropic Mycoplasma spp., Babesia spp., Anaplasma spp., Ehrlichia spp. and Hepatozoon spp. using microscopic examination, in-clinic ELISA testing and PCR assays. Dog-specific risk factors associated with the presence of CTBD pathogens were evaluated. RESULTS: Overall, 24.4% of the suspect group and 12.2% of the control group dogs were infected. The proportions of M. haemocanis, B. vogeli, A. platys, Candidatus Mycoplasma haematoparvum, and C. Mycoplasma haemobos were 7.1%, 5.0%, 3.8%, 1.7% and 0.4%, respectively. Dogs originating from the NT were 3.6-fold (95% confidence interval (CI) 1.51-8.62; P = 0.004) more likely to be infected with CTBD pathogens than those from SEQ. Male dogs were 2.3-fold (95% CI 1.17-4.80, P = 0.024) more likely to be PCR-positive to CTBD pathogens than female dogs. Dogs presenting with clinical signs consistent with CTBD and thrombocytopenia were more likely to be infected by CTBD pathogens (odds ratio 2.85; 95% CI 1.16, 7.02; P = 0.019). CONCLUSIONS: Haemotropic mycoplasmas were the most common tick-borne pathogen infecting client-owned dogs. Subclinical cases were common in dogs from the NT. Veterinary practitioners should be aware of the proportion of CTBD pathogens and the presenting features of clinical and subclinical disease in their area.

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.

Emergence of a 600-Hz buzz UP state Purkinje cell firing in alert mice.

Purkinje cell (PC) firing represents the sole output from the cerebellar cortex onto the deep cerebellar and vestibular nuclei. Here, we explored the different modes of PC firing in alert mice by extracellular recording. We confirm the existence of a tonic and/or bursting and quiescent modes corresponding to UP and DOWN state, respectively. We demonstrate the existence of a novel 600-Hz buzz UP state of firing characterized by simple spikes (SS) of very small amplitude. Climbing fiber (CF) input is able to switch the 600-Hz buzz to the DOWN state, as for the classical UP-to-DOWN state transition. Conversely, the CF input can initiate a typical SS pattern terminating into 600-Hz buzz. The 600-Hz buzz was transiently suppressed by whisker pad stimulation demonstrating that it remained responsive to peripheral input. It must not be mistaken for a DOWN state or the sign of PC inhibition. Complex spike (CS) frequency was increased during the 600-Hz buzz, indicating that this PC output actively contributes to the cerebello-olivary loop by triggering a disinhibition of the inferior olive. During the 600-Hz buzz, the first depolarizing component of the CS was reduced and the second depolarizing component was suppressed. Consistent with our experimental observations, using a 559-compartment single-PC model - in which PC UP state (of about -43mV) was obtained by the combined action of large tonic AMPA conductances and counterbalancing GABAergic inhibition - removal of this inhibition produced the 600-Hz buzz; the simulated buzz frequency decreased following an artificial CS.

'Cysticercosis storm' in feedlot cattle in north-west New South Wales.

OBJECTIVE: To investigate the cause of an outbreak of bovine cysticercosis (Taenia saginata) infection on a cattle property in north-western New South Wales (NSW). METHODS: Cystic lesions were detected in the muscles of cattle during routine meat inspection at slaughter. These lesions were confirmed to be cysticerci of T. saginata through histology and polymerase chain reaction (PCR). Data on cattle maintenance were obtained through interviews with feedlot owners and staff. A suspect feed supplement was investigated. RESULTS: Between 5 July to 13 December 2010, 390 feedlot cattle from north-western NSW were slaughtered in abattoirs in NSW and Queensland. Of these, 138 animals had been maintained exclusively in feedlot enclosures from 80 to 300 days. Bovine cysticercosis was discovered in 80 cattle (58%, 26 carcasses were condemned). Another 18 cattle spent 24 h in the feedlot before being moved onto pasture and 1 of them was found to be infected. During the 5 months following the initial outbreak, a further 275 cattle were slaughtered; 2 of 51 heifers retained in the feedlot for a further 100 days were infected. None of the 234 animals grazed exclusively on pasture on the property were infected. Bovine cysticercosis was confirmed through examination of histological sections of muscle lesions and PCR using DNA extracted from cysticerci. No eggs of T. saginata were recovered from the feed supplement using a standard flotation method. CONCLUSIONS: The source of infection arose from rations contaminated with human faeces. All possibilities for local contamination were eliminated during the investigation. The suspected source of infection was imported copra meal, which was used as a feed supplement.

Canine vector-borne disease pathogens in dogs from south-east Queensland and north-east Northern Territory.

OBJECTIVES: To determine the prevalence of canine vector-borne diseases (CVBD: Babesia spp., Anaplasma spp., Ehrlichia spp., haemotropic mycoplasmas and Hepatozoon) in Australian dogs; namely, dogs from pounds in south-east Queensland and an indigenous Aboriginal community in the north-east of the Northern Territory. DESIGN AND PROCEDURE: Blood samples were collected from 100 pound dogs and 130 Aboriginal community dogs and screened for the CVBD pathogens using polymerase chain reaction (PCR). All positive PCR products were sequenced for species confirmation. RESULTS: In total, 3 pound dogs and 64 Aboriginal community dogs were infected with at least one CVBD pathogen. Overall, B. vogeli was detected in 13 dogs, A. platys in 49, M. haemocanis in 23, Candidatus Mycoplasma haematoparvum in 3 and C. M. haemobos in 1 dog. Co-infections were detected in 22 Aboriginal community dogs. CONCLUSIONS: This study found B. vogeli, A. platys and haemotropic mycoplasma infections to be common in dogs in subtropical and tropical areas of Australia. This study also reports for the first time the prevalence and genetic characterisation of haemotropic mycoplasmas in dogs in Australia.

First report of a Trichinella papuae infection in a wild pig (Sus scrofa) from an Australian island in the Torres Strait region.

Multiple Trichinella species are reported from the Australasian region although mainland Australia has never confirmed an indigenous case of Trichinella infection in humans or animals. Wildlife surveys in high-risk regions are essential to truly determine the presence or absence of Trichinella, but in mainland Australia are largely lacking. In this study, a survey was conducted in wild pigs from mainland Australia's Cape York Peninsula and Torres Strait region for the presence of Trichinella, given the proximity of a Trichinella papuae reservoir in nearby PNG. We report the detection of a Trichinella infection in a pig from an Australian island in the Torres Strait, a narrow waterway that separates the islands of New Guinea and continental Australia. The larvae were characterised as T. papuae (Kikori strain) by PCR and sequence analysis. No Trichinella parasites were found in any pigs from the Cape York Peninsula. These results highlight the link the Torres Strait may play in providing a passage for introduction of Trichinella parasites from the Australasian region to the Australian mainland.

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.

Loop-mediated isothermal amplification test for Trypanosoma vivax based on satellite repeat DNA.

Trypanosoma vivax is major cause of animal trypanosomiasis and responsible for enormous economic burden in Africa and South America animal industry. T. vivax infections mostly run low parasitaemia with no apparent clinical symptoms, making diagnosis a challenge. This work reports the design and evaluation of a loop-mediated isothermal amplification (LAMP) test for detecting T. vivax DNA based on the nuclear satellite repeat sequence. The assay is rapid with results obtained within 35 min. The analytical sensitivity is ∼ 1 trypanosome/ml while that of the classical PCR tests ranged from 10 to 10(3)trypanosomes/ml. The T. vivax LAMP test reported here is simple, robust and has future potential in diagnosis of animal trypanosomiasis in the field.

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.

Prevalence of Cysticercus bovis in Australian cattle.

OBJECTIVE: The first national abattoir survey of Cysticercus bovis ('beef measles') in cattle was conducted in February 2008. METHODS: During the data collection period, 493,316 cattle were subjected to standard postmortem procedures, including incision of the masseter and heart muscles. On-site veterinarians were asked to record the location of any C. bovis cysts, as well as the National Livestock Identification System ear tag numbers of infected animals. Veterinarians were asked to submit samples for laboratory confirmation by histology and polymerase chain reaction testing. RESULTS: Of the 23 samples submitted, none was positive for C. bovis by either diagnostic method. CONCLUSIONS: Occasional, isolated diagnoses of beef measles are still made in most states of Australia, but since the last regional surveys were conducted 30 years ago, when the estimated prevalence was 50 to 200 per 100,000 cattle slaughtered, the parasite has become extremely rare.

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.

The visceromotor response to colorectal distention fluctuates with the estrous cycle in rats.

The existence of a sex difference in several chronic pain syndromes and the fluctuation of symptoms during the menstrual cycle strongly suggest sex hormones are involved in pain processing. The mechanisms underlying these changes are not well understood. Using the colorectal distention model in the rat, we previously reported a sex difference in the response to distention [Ji Y, Murphy AZ, Traub RJ (2006) Sex differences in morphine induced analgesia of visceral pain are supraspinally and peripherally mediated. Am J Physiol Regul Integr Comp Physiol 291:R307-R314] and that ovariectomy decreased the responses to distention while estrogen replacement reversed the decrease [Ji Y, Murphy AZ, Traub RJ (2003) Estrogen modulates the visceromotor reflex and responses of spinal dorsal horn neurons to colorectal stimulation in the rat. J Neurosci 23:3908-3915], suggesting estrogen increases visceral nociception. In the present study we tested the hypothesis that the visceromotor response to colorectal distention fluctuates with the estrous cycle. Three measurements (vaginal smears, uterine tube weight and plasma estrogen concentration) were used to determine the estrous phase. Comparison of the visceromotor response threshold and magnitude was made between proestrus and metestrus/diestrus. Our experiment demonstrated that the distention threshold was significantly lower in proestrus (median: 15 mm Hg) as compared with metestrus/diestrus (median: 25 mm Hg); and the magnitude of the visceromotor response to graded intensities of colorectal distentions (20, 40, 60, 80 mm Hg) was significantly higher in proestrus. The results indicate that the visceromotor response fluctuates with estrous phase, providing evidence for endogenous estrogen modulation of visceral nociceptive processing that could contribute to sex differences.

Blastocystis: subtyping isolates using pyrosequencing technology.

Blastocystis is a prevalent single-celled enteric parasite of unresolved clinical significance. Efforts based on molecular methodologies to establish whether pathogenicity is linked to specific isolates of the genetically diverse genus of Blastocystis have been scarce and so far yielded ambiguous results which can be difficult to interpret. To alleviate some of the problems related to unravelling the molecular epidemiology of Blastocystis infections we developed and evaluated a simple and high-throughput sequence analysis (SQA) pyrosequencing technique based on the detection of genotype-specific nucleotide polymorphisms in the 18S small subunit rRNA gene for a rapid and cost-effective post-PCR screening of Blastocystis genotypes. The method was effectively capable of genotyping 48/48 isolates positive by nested PCR in approximately one hour, and in 94% of the cases the isolate detected by PCR and pyrosequencing was also detected by one of two different PCR assays with subsequent dideoxy sequencing.

A case of mistaken identity--reappraisal of the species of canid and felid hookworms (Ancylostoma) present in Australia and India.

This study serves to clarify the current status of canid and felid Ancylostoma species present in Australia. The morphological identification of A. ceylanicum from cats for the first time in Townsville, Australia, appears to be in error, together with the genetic markers provided for the species. Morphological and genetic data presented herein provide strong evidence that the hookworms from cats in Towsville are not A. ceylanicum as previously identified (i.e. the first report of this species in Australia), but are A. braziliense. Therefore the subsequent genetic markers established for A. ceylanicum in subsequent molecular studies based on these Townsville specimens should also be attributed to A. braziliense. Based on this information, a study of canine hookworm species present in northern India is also in error and it is apparent that the hookworms found in this region are those of A. ceylanicum. The distribution of A. braziliense and A. ceylanicum in the Americas and Asia Pacific region is discussed together with the importance of combining parasite morphology with genetic data for parasite diagnosis in epidemiological studies.