Genotyping of European Toxoplasma gondii strains by a new high-resolution next-generation sequencing-based method.

PURPOSE: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. METHODS: T. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. RESULTS: Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing. CONCLUSION: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.

Preparedness for chemical crisis situations: experiences from European medical response exercises.

OBJECTIVE: This study investigated how European first responders and hospital personnel, along with CBRN experts, approach an overwhelming surge situation after a chemical incident. Surge capacity and capability bottlenecks were discussed. MATERIALS AND METHODS: Two chemical warfare agent (CWA) scenarios were developed: in the first, a nerve agent was released indoors; in the second, there was an outdoor explosion containing a blister agent. CBRNE experts, first responders and hospital specialists from across Europe participated in a two-day table-top exercise to discuss pre-hospital and hospital CBRNE preparedness, triage, surge capacity and communication issues. This was followed by a medical response exercise at a level 2 Emergency Department in Italy. RESULTS: Several surge capacity challenges and lessons were identified. Critical resources were rapidly exhausted and sourcing from national/international medical stockpiles was not feasible in the time critical scenarios. Secondary contamination in the blister agent scenario was considered plausible and hospitals are currently unprepared for this situation. The medical response exercise highlighted further training needs. CONCLUSIONS: The majority of the lessons are not new and have been reported in North American studies. However, this study is the first to describe these CWA challenges from a European perspective. Medical facilities across the region should consider these lessons to evaluate and improve their surge capacity, capability and response.

Forest classes and tree cover gradient: tick habitat in encroached areas of southern Norway.

Forest, in particular deciduous forest, is a key element in determining areas with a high probability of tick presence. The way forest is generally monitored may be ill suited to some landscapes where Ixodes ricinus is found, as forest is usually characterised using crisp land cover classes. However, tree vegetation can be found outside of forests and continuous gradations of tree density can be found in a variety of landscapes. In this paper we investigate the probability of tick presence in southern Norway using landscape description based both on land cover classes and continuous data describing the tree cover fraction. Both perspectives on the landscape are significant in the logistic model, indicating that the usual approach based solely on land cover classes may not be comprehensive enough in capturing tick habitat, and characterising the landscape with variables focused on single specific elements may be insufficient.

Dickkopf-3 is upregulated in osteoarthritis and has a chondroprotective role.

OBJECTIVE: Dickkopf-3 (Dkk3) is a non-canonical member of the Dkk family of Wnt antagonists and its upregulation has been reported in microarray analysis of cartilage from mouse models of osteoarthritis (OA). In this study we assessed Dkk3 expression in human OA cartilage to ascertain its potential role in chondrocyte signaling and cartilage maintenance. METHODS: Dkk3 expression was analysed in human adult OA cartilage and synovial tissues and during chondrogenesis of ATDC5 and human mesenchymal stem cells. The role of Dkk3 in cartilage maintenance was analysed by incubation of bovine and human cartilage explants with interleukin-1ß (IL1ß) and oncostatin-M (OSM). Dkk3 gene expression was measured in cartilage following murine hip avulsion. Whether Dkk3 influenced Wnt, TGFß and activin cell signaling was assessed in primary human chondrocytes and SW1353 chondrosarcoma cells using qRT-PCR and luminescence assays. RESULTS: Increased gene and protein levels of Dkk3 were detected in human OA cartilage, synovial tissue and synovial fluid. DKK3 gene expression was decreased during chondrogenesis of both ATDC5 cells and humans MSCs. Dkk3 inhibited IL1ß and OSM-mediated proteoglycan loss from human and bovine cartilage explants and collagen loss from bovine cartilage explants. Cartilage DKK3 expression was decreased following hip avulsion injury. TGFß signaling was enhanced by Dkk3 whilst Wnt3a and activin signaling were inhibited. CONCLUSIONS: We provide evidence that Dkk3 is upregulated in OA and may have a protective effect on cartilage integrity by preventing proteoglycan loss and helping to restore OA-relevant signaling pathway activity. Targeting Dkk3 may be a novel approach in the treatment of OA.

Cross-border transport of rescue dogs may spread rabies in Europe.

Harmonisation of regulations in the European Union and the European Economic Area, as of January 1, 2012, has led to an increase in the number of rescue dogs imported to Norway from Eastern European countries, in particular Romania. Today the only requirements for dogs entering Norway are rabies vaccination and prophylactic Echinococcus multilocularis treatment. The aim of this study was to investigate the antibody levels to rabies virus in vaccinated rescue dogs and to examine if the dogs had sufficient antibody response according to the recommended titre ≥0.5 IU/ml by the World Organisation for Animal Health (OIE). A significant proportion (53%, 95% CI (41% to 65%)) of imported rescue dogs from Eastern Europe were found to have inadequate titres after rabies vaccination. Moreover, 41 per cent of the dogs had antibody levels below or equal to 0.2 IU/ml, and among these, 14 dogs had titres ≤0.1 IU/ml, which is considered negative in the fluorescent antibody virus neutralisation assay. This study indicates that the present regulation increases the risk of introducing rabies from member states where rabies is still prevalent to countries considered free from rabies.

European pet travel: misleading information from veterinarians and government agencies.

Inter-country travel of companion animals provides an opportunity for introduction of zoonotic pathogens, such as rabies virus and Echinococcus spp. Regulations are in place to control this threat, but Schengen Agreements mean that border controls between some European countries are minimal, and animals may enter countries without any checks that they have been appropriately treated. Veterinarians provide an important source of information for people intending to travel with their pets. We conducted a telephone survey to investigate provision of correct advice to someone intending to travel with their dog to Norway. Mainland Norway is considered free of both rabies and E. multilocularis and is a signatory to the Schengen Agreement. Ten randomly selected veterinary clinics were surveyed in Austria, Belgium (Wallonia), Finland, France, Germany, Norway, Sweden, Switzerland and United Kingdom. The information provided was scored as correct, incorrect or incomplete. The information provided by secondary information sources (website or government agency), which the clinic had referred the caller to, was also assessed (correct, incorrect, incomplete). Whilst the majority of clinics provided appropriate information regarding rabies, many clinics did not provide correct information regarding treatment for E. multilocularis. Less than one in 10 clinics provided the correct information regarding both pathogens directly at the time of calling. The correct information was obtained, once taking into account secondary sources, just 62% of the time. Countrywise, most clinics in Finland provided correct advice, either directly or indirectly via referring the caller to another source, whilst the majority in Belgium, Germany and France did not. The apparent paucity of readily accessible, correct advice for owners intending to travel with their dogs is concerning. The compulsory treatment regulations are only as good as the checks that ensure compliance, and this is also lacking in some countries.

Phenelzine produces subsensitivity to nicotine.

1. The authors attempted to detect a possible effect of treatment with phenelzine on a physiological response to nicotine in the rat. 2. Positive findings in an animal model suggest the feasibility of more complicated experiments in animals and the possibility of studies involving human subjects. 3. Treatment of Sprague Dawley rats (n = 10) with phenelzine sulfate (15.0 mg/kg ip) every 48 hours for 14 days was associated with a 73.3% decrease in the hypothermic response to nicotine. 4. Treatment with phenelzine did not enhance the rate of elimination of nicotine. 5. The authors discuss a possible relationship between changes in nicotinic mechanisms and the therapeutic actions of drugs used to treat affective illness.

A nicotinic receptor antagonist enhances the hypothermic response to a muscarinic agonist.

1. Chronic treatment with amitriptyline produces supersensitivity to the hypothermic effects of the muscarinic agonist oxotremorine. 2. Chronic treatment with amitriptyline also produces supersensitivity to the hypothermic effects of nicotine. 3. Oxotremorine and other naturally occurring muscarinic agonists are also nicotinic agonists. 4. Chronic treatment with amitriptyline produces time-dependent and reversible supersensitivity to the hypothermic effects of nicotine. 5. The authors assessed the possibility that the development of supersensitivity to oxotremorine following chronic treatment with amitriptyline is related to an effect of this antidepressant on a nicotinic mechanism. 6. A nicotinic receptor antagonist would blunt (though not necessarily eliminate) enhanced sensitivity to the thermic effects of oxotremorine if the nicotinic effects of the latter are significant. 7. The simultaneous administration of mecamylamine (a peripherally and centrally active nicotinic receptor antagonist) greatly augments (rather than blunts) the hypothermic response to oxotremorine. 8. The data suggest that the oxotremorine may activate a nicotinic mechanism counterbalancing its effect on a muscarinic mechanism. 9. This is consistent with previously published reports that the activation of nicotinic and muscarinic mechanisms can exert opposite effects.

Chronic treatment with amitriptyline produces subsensitivity to the hypothermic effects of yohimbine.

1. Two but not one week of treatment with amitriptyline (AMI) produces subsensitivity to the hypothermic effects of the alpha 2 antagonist yohimbine. 2. Subsensitivity persisted for the three weeks during which it was measured following the discontinuation of AMI. 3. Two weeks of twice daily injections of saline did not alter the thermic response to this agent. 4. The results support other data indicating tricyclic antidepressants (TCAs) subsensitize alpha 2 mediated thermic changes and are consistent with reports that TCAs down-regulate alpha 2 receptors.

Desipramine subsensitizes nicotinic mechanism involved in regulating core temperature.

Desipramine HCl, 10 mg/kg i.p. twice daily, produced subsensitivity to the hypothermic effects of nicotine, 1 mg/kg, after 1 and 2 weeks of treatment in male Sprague-Dawley rats, Phenelzine sulfate, fluoxetine HCl, and bright artificial light produced the same effect. The capacity of three chemically distinct classes of anti-depressants and bright artificial light (a treatment for seasonal depression) to produce this result suggests that effects on nicotinic mechanisms may be involved in the mechanism of action of these treatments.

Fluoxetine subsensitizes a nicotinic mechanism involved in the regulation of core temperature.

Fluoxetine HCl, 10 mg/kg ip, twice daily produced subsensitivity to the hypothermic effects of nicotine (base), 1 mg/kg ip, after 1 (p less than 0.02) and 2 (p less than 0.002) weeks of treatment. Phenelzine sulfate, desipramine HCl and bright artificial light produced the same effect. The capacity of three chemically distinct classes of antidepressants and bright artificial light (a treatment for seasonal depression) to produce this result suggests that effects on nicotinic mechanisms may be involved in the mechanism of action of these treatments.

The economics of parasitic diseases: research priorities.

Parasitic diseases are primarily diseases of poverty. At serious risk are individuals, communities and countries least able to afford the costs of treatment or prevention. In turn, economic development projects which aim to increase income levels may lead to negative results because of increased transmission of parasitic diseases often results. In attempting to analyse the economic consequences of parasitic diseases and the economics of their control, economists have usually relied on the tools of cost-benefit analysis, cost-effectiveness analysis and financial analysis. These efforts are briefly reviewed in the paper. The results have been subject to considerable criticism because of conceptual and methodological problems. For example, most studies have not taken into account the epidemiology and natural history of the disease in estimating the associated economic losses, thereby leading to inappropriate conclusions. The UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases through its Social and Economic Research Scientific Working Group (SER-SWG) is promoting research on the economics of parasitic diseases. In an attempt to improve the usefulness and reliability of such studies, conceptual and methodological approaches have been suggested which are reported on here. To improve the research design of these projects, a conceptual framework is proposed which consists of four components: (1) baseline social, cultural and economic conditions influencing disease transmission; (2) resources already invested in the health system and investments in other related sectors such as agriculture, housing, water supply and sanitation; (3) health consequences resulting from (1) and (2); and (4) social and economic consequences resulting from (3). A key concern in relation to the framework is to determine the most useful basis for linking results from studies of one component to another so as to analyse more systematically the impacts of disease on individuals and society. Such studies are inherently interdisciplinary and close collaboration of economists with medical scientists and disease control programme staff is needed to ensure completeness and reliability of input data and results. Results from these studies could be used to inform national decision-makers about the social and economic consequences of the parasitic diseases and their control and, thus, should strengthen support for increased investment to reduce the parasitic disease burden in developing countries.

Potassium evoked catecholamine release from the nucleus tractus solitarius in vitro.

The release of endogenous catecholamines (CA) from rat brain slices containing the nucleus tractus solitarius (NTS) was measured using a sensitive radioenzymatic assay. KCl (35 to 75 mM) induced a dose-related increase in norepinephrine (NE) release. Dopamine (DA) release was maximal with 50 mM KCl. An increase in epinephrine (E) release was only observed with 75 mM CKl. NE and E release was totally calcium-dependent whereas DA release was only partially calcium-dependent. Subsequent administrations of KCl released less CA. The calcium dependency of the KCl induced released of E, NE, and DA suggests a neurotransmitter function in the NTS for these CA. A difference in storage sites and/or mechanisms may be responsible for the observed differences in sensitivity to KCl and to extracellular calcium.

Effect of prophylactic antibiotics in acute nonperforated appendicitis: a prospective, randomized, double-blind clinical study.

A prospective, randomized, double-blind clinical study was performed to determined the efficacy of short-term (24 hr) perioperative antibiotics in preventing septic complications after emergency appendectomy for nonperforated appendicitis. The patients were stratified into three clinical arms: Group I (placebo, n = 45), Group II (cefamandole, n = 46) and Group III (cefamandole plus carbenicillin, n = 45). The three groups of patients were similar in regard to age, sex, duration of operation and pathologic classification of the appendix. The overall incidence of infection in the study was 5.1%. The infection rates in Groups II (2.2%) and III (0%) were significantly lower than Group I (placebo) (13.3%), (p less than 0.05). No difference was observed between cefamandole alone and cefamandole plus carbenicillin. Average postoperative hospital days per patient for each group was: Group I - 3.8 days; Group II - 2.9 days; Group III - 3.1 days. Cost analysis of hospitalization including cost of prophylactic antibiotics revealed a $247.99 per patient saving for Group II versus Group I and $95.53 for Group III versus Group I. Systemic prophylactic antibiotics can successfully reduce septic complications after appendectomy for nonperforated appendicitis, and a single drug (cefamandole) directed at the facultative pathogens is as effective as double drug therapy, which includes specific anaerobic coverage.

Carotid artery stenosis - hemodynamic significance and clinical course.

Two hundred fifteen patients with a history of either stroke, transient ischemic attack (TIA), or asymptomatic carotid bruit underwent noninvasive carotid artery testing using oculopneumoplethysmography. Of patients with hemodynamically significant stenosis, 51 (40.8%) underwent endarterectomy, and 74 (59.2%) were treated nonoperatively. The incidence of stroke in the nonoperated group was 12/74 (16.2%) compared with only 1/51 (1.9%) in the operated group. Similarly, recurrent TIA occurred in 29/74 (39.2%) of the nonoperated group vs 9/51 (17.6%) of the operated. In nonhemodynamically significant carotid stenosis, the risk of cerebrovascular death and stroke was exceedingly low: 2/90 (2.2%). Patients with hemodynamically significant stenosis treated nonoperatively have a greater risk of cerebrovascular death, stroke, and TIA than patients treated with carotid endarterectomy.