Discrimination of Culicoides midge larvae using multiplex polymerase chain reaction assays based on DNA sequence variation at the mitochondrial cytochrome C oxidase I gene.

The recent spread of Bluetongue disease in northwestern Europe has indicated the ability of Palaearctic Culicoides species to vector the disease. Because the different midge species vary in their ability to harbor and transmit the Bluetongue virus, quick and reliable identification is necessary to resolve the species composition of midge communities, both adult and larval, at any place at any given time point. Given that morphological identification of Culicoides species is problematic, we developed three multiplex polymerase chain reaction (PCR) assays that facilitate high-throughput analysis of midge specimens. One assay distinguishes between species of the so-called Culicoides obsoletus s.l. complex (including C. dewulfi), whereas two assays facilitate differentiation of species of the Culicoides pulicaris s.l. complex. These assays yield two PCR products: one species-specific and one generic band. We show the application of the assays in the analysis of Culicoides larvae from three different farms in northeast Scotland.

Identification of human-derived volatile chemicals that interfere with attraction of the Scottish biting midge and their potential use as repellents.

The Scottish biting midge, Culicoides impunctatus (Diptera: Ceratopogonidae), is a major pest in Scotland, causing a significant impact to the Scottish tourist and forestry industries. C. impunctatus is a generalist feeder, preferring to feed on large mammals, and is notorious for its attacks on humans. Until now, there was anecdotal evidence for differential attraction of female host-seeking C. impunctatus to individual human hosts, and the mechanism for this phenomenon was unknown. Using extracts of human odor collected by air entrainment, electroantennogram recordings to identify the physiologically active components, followed by behavioral assays, we show, for the first time, the differential attraction of female C. impunctatus to human odors and the chemical basis for this phenomenon. Certain chemicals, found in greater amounts in extracts that cause low attractiveness to midges, elicit a repellent effect in laboratory assays and repellency trials in the field. Differences in the production of these natural human-derived compounds could help to explain differential "attractiveness" between different human hosts. A mixture of two compounds in particular, 6-methyl-5-hepten-2-one and geranylacetone [(E)-6,10-dimethylundeca-5,9-dien-2-one], showed significant repellency (87, 77.4, 74.2, and 31.6% at hours 0, 1, 2, and 3, respectively) in the field and have the potential to be developed as novel repellents.

Phylogenetic analysis indicates that Culicoides dewulfi should not be considered part of the Culicoides obsoletus complex.

Analysis of DNA sequence data has proven invaluable for defining the relationships among taxa, as well as resolving their evolutionary histories. Here, we analyzed DNA sequence variation of one mitochondrial gene (COI) and two nuclear regions (ITSI and II) to clarify the phylogenetic position of Culicoides dewulfi, a midge species widely spread in Europe and a suspected vector for bluetongue virus. Various authors have described C. dewulfi either as part of the Culicoides obsoletus sensu lato complex or as a separate taxonomic group. A maximum likelihood phylogeny, based upon an optimal model of sequence evolution, placed C. dewulfi outwith the C. obsoletus s.l. complex. Shimodaira-Hasegawa test highlighted that this topology was significantly more likely than any topology that placed C. dewulfi anywhere else in the phylogeny. As such, C. dewulfi should not be considered part of the C. obsoletus s.l. complex and instead be treated as a separate group, phylogenetically close to the classical Old World vector C. imicola.

Azadirachtin disrupts formation of organised microtubule arrays during microgametogenesis of Plasmodium berghei.

Transmission of malaria parasites from vertebrate blood to the mosquito vector depends critically on the differentiation of the gametocytes into gametes. This occurs in response to environmental stimuli encountered by the parasite in the mosquito bloodmeal. Male gametogenesis involves three rounds of DNA replication and endomitosis, and the assembly de novo of 8 motile axonemes. Azadirachtin, a plant limnoid and insecticide with an unkown mode of action, specifically inhibits the release of motile gametes from activated microgametocytes but does not inhibit growth and replication of a sexual blood stages. We have combined confocal laser scanning microscopy and transmission electron microscopy to examine the effect of azadirachtin on the complex reorganisation of the microtubule cytoskeleton during gametogenesis in Plasmodium berghei. Neither the replication of the genome nor the ability of tubulin monomers to assemble into microtubules upon gametocyte activation were prevented by azadirachtin. However, the drug interfered with the formation of mitotic spindles and with the assembly of microtubules into typical axonemes. Our observations suggest that azadarachtin specifically disrupts the patterning of microtubules into more complex structures, such as mitotic spindles and axonemes.

Semiochemical strategies for sea louse control: host location cues.

The development of behavioural bioassays and electrophysiological recording techniques has enabled the role of semiochemicals to be investigated for the first time in Lepeophtheirus salmonis (Krøyer). Adult male sea lice L salmonis were activated by, and attracted to, salmon-conditioned water (SCW) and SCW extract. Non-host fish odours, turbot-conditioned water (TCW) and turbot-conditioned water extract (TCW extract), elicited activation but no attraction in the lice. Solid-phase extraction techniques were developed to extract low molecular weight components of fish odours, and biological activity was shown to be confined to these. Electrophysiological activity from aesthetascs in the first antennae was found and neural responses to odours were recorded from the antennal nerves. Preliminary experiments on individual chemicals (isophorone, 1-octen-3-ol) linked to salmon revealed behavioural activation and electrophysiological responses in adult male L salmonis. Isophorone was shown to be significantly attractive to sea lice when placed in a slow-release system in a perfused tank of seawater in a choice situation. Proof of concept has been established for successfully extracting odour cues from seawater, analysing their biological activity and applying these to slow-release technologies for field trapping of lice. Future work involving linked GC-MS techniques using behaviour, electrophysiological responses, and organolepsis will establish further host location cues specific to Atlantic salmon.

Counselling: filling a gap in general practice.

A counsellor worked for 1 year in three practices in a rural area where there was previously no practice-based counselling. The service was evaluated, using a range of methods, in order to inform general practitioners and policy-makers about the demand for counselling, where it fits with other services, its potential value and how to organise and audit the service efficiently. Five kinds of information were collected: administrative data; patients' views; well-being scores; GPs' perceptions of individual patients; and interviews with the counsellor, GPs and other primary care staff by an independent researcher. All the GPs used the service, referring 131 people. The most common reason for referral was 'relationship difficulties'. There were improvements in patients' well-being, self-awareness and coping skills, and high satisfaction among GPs and patients. Communication with other services was seen to improve. The counselling service was found to fill a gap by addressing the needs of a substantial group of patients for whom psychiatric care was inappropriate.

The development of an approach to evaluate NHS manager's competence in medical informatics.

This paper describes the research and development of Checkpoint, a self-assessment instrument. It outlines how the instrument has helped managers in the NHS identify their strengths and weaknesses with respect to their management and use of informatics. The paper presents trends and feedback from the use of Checkpoint and the implications for future education and training of managers in the NHS.

Building an information culture: the top team.

This paper describes a learning process, used by the NHS, which aims to improve the management of information in NHS organizations by enabling the top team to develop a more strategic view of information management and technology in line with the Management in the 1990s approach.

Thresholds for treatment in cataract surgery.

BACKGROUND: The aim of the study was to identify the criteria used by ophthalmologists when assessing patients who may require cataract surgery, examine the extent of variation in their application and explore their relationship with current patterns of supply and demand. METHODS: This was a descriptive study involving semi-structured interviews with ophthalmologists, examination of hospital clinical records, and analysis of routinely available data on waiting times and hospital activity. The setting was ophthalmology units within the Northern Region of England. The subjects were 27 consultant ophthalmologists and 160 patients undergoing cataract extraction. RESULTS: There was agreement amongst ophthalmologists on the criteria used to select patients for treatment, and on the visual acuity level at which they would usually recommend surgery. All assess the degree of handicap resulting from cataract; most consider this more important than visual acuity. Over half of the patients were over 75 years old; two-thirds were women. Median visual acuity at listing was 6/36, but over 40 per cent were 6/60 or worse. Visual acuity at treatment was not recorded for 32 per cent of patients. Wide variation in visual acuity at listing existed between both units and consultants, and for both the affected and other eye. Second extractions may be performed at a better level of visual acuity than for first extractions. Median visual acuity at listing was significantly correlated with total waiting times for individual consultants. Lower cataract extraction rates are associated with long waiting times and poorer visual acuity at listing. CONCLUSIONS: There is considerable unmet need for cataract treatment within the Northern Region and significant variation in the current meeting of needs and demands. It may be that more needs could be met simply by changing referral and treatment patterns without increasing total service activity. Although visual acuity is a reasonably objective measure in routine use, the level of visual handicap is more important, and its assessment is more subjective; development of a standard method to assess this could help in producing guidelines for patient referral and selection. Audit of treatment thresholds could allow a better matching of service provision to population needs.

Effect of guidelines on management of head injury on record keeping and decision making in accident and emergency departments.

OBJECTIVE: To compare record keeping and decision making in accident and emergency departments before and after distribution of guidelines on head injury management as indices of implementation. DESIGN: Before (1987) and after (1990) study of accident and emergency medical records. SETTING: Two accident and emergency departments in England. PATIENTS: 1144 adult patients with head injury in department 1 (533 in 1987, 613 in 1990) and 734 in department 2 (370, 364 respectively). MAIN MEASURES: Recording of relevant symptoms and signs as determined in the guidelines; presence of, indications for, and rates and appropriateness of skull x ray examination and admission. RESULTS: The median number of guidelines variables recorded for all study periods ranged from 7 to 9 out of a possible maximum of 27. For key decision making variables the presence or absence of penetrating injury was least likely to be recorded (< or = 1%) and that of loss of consciousness most likely (> or = 75%). Altogether, the proportion of patients receiving skull x ray examination or admitted varied from 25%-60% and 7%-23% respectively; overall, 69% (1280/1856) and 64% (1177/1851) of patients were managed appropriately. However, no consistent change occurred in the departments between the study periods. For instance, in department 1 the proportion of appropriate x ray examinations rose significantly after distribution of the guidelines (from 61% (202/330) to 73% (305/417)) and appropriate decisions on whether to x ray or not also rose (from 65% (340/522) to 72% (435/608)). There was no significant change in department 2, although the proportion of appropriate admissions fell (from 33% (55/166) to 15% (19/130)). CONCLUSIONS: Recording practice and decision making were variable and had not consistently improved after dissemination of the guidelines. Strategies are required to ensure effective implementation of guidelines.

Identification of bloodmeals of the Scottish biting midge, Culicoides impunctatus, by indirect enzyme-linked immunosorbent assay (ELISA).

An indirect enzyme-linked immunosorbent assay (ELISA) has been developed to identify the bloodmeal sources of Culicoides impunctatus Goetghebuer. Nightly light trapping in Scotland, between April and September 1992, caught a total of 344 blood-engorged female C. impunctatus, all in May, June and July. The insects were stored in 70% ethanol and tested 5-7 months later. Bloodmeal sources were positively identified for 246 (71.5%). Bovine hosts were most common (38.4%), followed by deer (23.0%) and sheep (9.9%). Only one midge had fed on human blood. Relatively freshly fed (< 24 h), fully engorged females gave the clearest results. The wider applications of the technique to the study of Culicoides host preferences is discussed.

An audit of distribution and use of guidelines for management of head injury.

Ensuring effective distribution of guidelines is an important step towards their implementation. To examine the effectiveness of dissemination of a guidelines card on management of head injury and determine its usefulness to senior house officers (SHOs), a questionnaire survey was performed in May 1990, after distribution of the cards in induction packs for new doctors and at postgraduate lectures and displaying the guidelines in accident and emergency departments and wards. A further survey, in March 1992, assessed the impact of modifying the distribution. All (175) SHOs working in general surgery, accident and emergency medicine, orthopaedics, and neurosciences on 1 February 1990 in 19 hospitals including two neurosurgical units in Northern region were sent self completion questionnaires about awareness, receipt, use, and perceived usefulness of the guidelines. 131 of 163(80%) SHOs in post responded (median response from hospitals 83% (range 50%-100%)). Over three quarters (103, 79%) of SHOs were aware of the guidelines and 82(63%) had ever possessed a guidelines card. Only 36(44%) acquired the card in the induction pack. 92%(98/107) found them useful and 81% (89/110) referred to them to some extent. Owning and carrying the card and referring to guidelines were associated with departmental encouragement to use the guidelines. Increasing the displays of guidelines in wards and departments and the supply of cards to consultants in accident and emergency medicine as a result of this survey did not increase the number of SHOs who received cards (52/83, 63%), but more (71/83, 86%) were aware of the guidelines. The guidelines were welcomed by SHOs and used in treating patients with head injury, but their distribution requires improvement. Increased use of the guidelines may be achieved by introducing other distribution methods and as a result of encouragement by senior staff.

Morphology of the antennae of two species of biting midge: Culicoides impunctatus (Goetghebuer) and Culicoides nubeculosus (Meigen) (Diptera, Ceratopogonidae).

Scanning and transmission electron microscopy of the antennae of Culicoides impunctatus and Culicoides nubeculosus show that males and females share five sensillum types. Sensilla chaetica resemble mechanoreceptors, each innervated by a single neurone whose dendrite terminates distally in a tubular body: the arrangement of sensilla on male antennae suggests that females are located by sound. The antennae have both sharp- and blunt-tipped sensilla trichodea, sharp-tipped sensilla on only the distal third and blunt-tipped sensilla on all subsegments. These sensilla are typical of olfactory receptors, having multiporous walls and being innervated by a number of neurones with bifurcating dendrites ascending the hair shafts. Sensilla basiconica occur on the distal five subsegments of the female antenna and the distal three subsegments of the male antenna. Sensilla coeloconica always occur on subsegment one and sometimes on a number of other subsegments, depending on sex and species. Both basiconic and coeloconic sensilla have double walls and unbranched dendrites and may be either olfactory or thermo- and/or hygroreceptors. All antennae except those of male C. impunctatus antennae have sensilla ampullacea, apparently deep-seated olfactory or thermoreceptors. Small peg sensilla fitting the description of contact chemoreceptors occur only at the tip of the male antenna.