Analysis of intrahost variation in Venezuelan equine encephalitis virus reveals repeated deletions in the 6-kilodalton protein gene.

RNA viruses exist as a spectrum of mutants that is generated and maintained during replication within the host. Consensus sequencing overlooks minority genotypes present in the viral sample that may impact the population's phenotype. In-depth sequencing of an original field isolate of subtype IE Venezuelan equine encephalitis virus (VEEV) demonstrated the presence of multiple deletions within the 6,000-molecular-weight (6K) protein gene. Using in vitro and in vivo experiments, similar deletions were generated in an additional VEEV strain originating from an infectious cDNA clone. Time course experiments demonstrated that the deletions are produced during acute infection although not until 24 h postinfection. Molecular clones containing some of these deletions were generated, and although the larger deletions appear to be noninfectious, viruses with the smaller deletions were viable and formed small plaques. Serial passages provided no evidence that these deletion mutants function as defective interfering particles. Furthermore, since wild-type infections generally occur at a low multiplicity of infection, it is unlikely that these deletions are propagated in natural transmission cycles. However, they could affect pathogenesis at later stages of infection. Because they are ubiquitously generated both in vivo and in vitro, further investigation is warranted to understand the generation of these deletions and their significance for disease.

The effect of skin allografting on the equine endometrial cup reaction.

This research tested the hypothesis that immunological sensitization of mares by skin allografting, followed by the establishment of pregnancy using semen from the skin-graft donor, would give rise to secondary immune responses to the developing horse conceptus, resulting in an earlier demise of the fetally derived endometrial cups. Maiden mares received skin allografts from a stallion homozygous for Major Histocompatibility Complex (MHC) antigens and/or equivalent autografts and were subsequently mated to the skin-graft donor stallion during the next two breeding seasons. Mares that had been immunologically primed to the foreign MHC class I antigens of the skin-graft donor stallion developed strong secondary antibody responses early in their first pregnancies, whereas autografted mares made weak primary antibody responses in their first pregnancies and strong secondary responses in their second pregnancies. In contrast, histological examination of the endometrial cups after surgical pregnancy termination at Day 60 of gestation revealed no discernible differences between allografted and autografted mares, and there were no significant differences in the concentrations and/or duration of secretion of the endometrial cup-specific hormone, equine chorionic gonadotrophin (eCG), between allografted and autografted mares, nor in either group between first and second pregnancies. The vigorous antibody response observed in the pregnant allografted mares supported the first part of our hypothesis, providing evidence of systemic immunological priming. However, there was a lack of an equivalent heightened cellular response to the endometrial cups. These findings provided strong evidence for an asymmetric immune response to the conceptus, characterized by strong humoral immunity and a dampened cellular response.

Dead space and paediatric anaesthetic equipment: a physical lung model study.

Untested assumptions have been made with regard to functional dead space in facemasks, filters and breathing systems used in children for the administration of inhalation anaesthesia. Total functional dead space was measured in various combinations of this equipment applied to a spontaneous ventilation lung model with parameter settings appropriate for infants of 7-8 kg. We found that functional dead space was too large to allow for spontaneous ventilation of the lungs when a breathing filter was fitted. There was minimal relationship between size of the facemask and functional dead space; however, the provision of 22-mm female inlets to facemasks achieved proportionately less functional dead space than with 15-mm male inlets. Regardless of the apparatus used and the magnitude of the dead space, the leak induced when a poorly fitting facemask was used dramatically reduced the dead space of the breathing system - to near optimal conditions - by moving the alveolar gas elimination point to within the facemask itself.

Ectopic transplantation of equine invasive trophoblast.

A system for transplanting invasive equine trophoblast (i.e., chorionic girdle) to ectopic sites has been developed as a means to study the differentiation of this tissue and to assess maternal immune responses to the conceptus tissue in a site outside the uterus. Chorionic girdle was isolated from Day 33 to 34 conceptuses and surgically placed into the vulvar mucosa or subdermal skin of recipient mares. Biopsy specimens of the graft sites for immunohistochemical staining were taken at weekly or biweekly intervals after grafting. Serum samples were collected from each recipient and tested for antibody to donor major histocompatibility complex (MHC) class I antigens using the lymphocyte microcytotoxicity assay. Transplanted trophoblast cells expressed differentiation markers associated with invading chorionic girdle and endometrial cup cells. The transplanted trophoblast cells were also labeled by an antibody to eCG. Strong cellular and humoral immune responses to the transplanted tissue were mounted by the recipients, similar to those occurring during normal equine pregnancy. Despite these responses, the invasive trophoblast transplants survived for at least 28 days after grafting and downregulated MHC class I antigens, as do the mature endometrial cup cells in equine pregnancy. These findings suggest that invasive equine trophoblast has the capacity to differentiate fully in equine nonuterine tissues, and that it can evade maternal immune responses independent of the physiological state of pregnancy and in sites other than the uterus.

Antibacterial properties of a silver chloride-coated nylon wound dressing.

OBJECTIVE: A silver chloride-coated nylon wound dressing (Ag-WD) was evaluated in vitro for antimicrobial activity against five common equine wound pathogens. STUDY DESIGN: Bacterial susceptibility study. SAMPLE POPULATION: Equine wound pathogens: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus equi subspecies zooepidemicus, and Staphylococcus aureus. METHODS: An inoculum of each pathogen was incubated directly with Ag-WD and quantitated after 24 to 48 hours of incubation. To determine if bactericidal activity of Ag-WD was contact dependent, an inoculum of E. coli and Staphylococcus aureus was incubated separately from Ag-WD by a filter and quantitated after 18 hours of incubation. Inductively coupled plasma emission spectrometry (ICP) determined the silver concentration of Mueller-Hinton broth containing Ag-WD after 24 hours of incubation. To establish if the rate of bacterial killing by Ag-WD differed from a constant silver concentration, pathogens were exposed to a silver concentration of 6.45 microg/mL and quantitated after 18 hours. RESULTS: Direct exposure to Ag-WD significantly reduced bacterial numbers after 15 minutes for K. pneumoniae, 30 minutes for E. coli, 1 hour for P. aeruginosa, and 2 hours for S. equi subspecies zooepidemicus and Staphylococcus aureus. Indirect exposure to Ag-WD resulted in > or =99.9% and > or =90% kill of the inoculum doses of E. coli at 2 hours and Staphylococcus aureus at 18 hours, respectively. Incubation of the pathogens at the constant silver concentration resulted in bacterial killing rates similar to those obtained by incubation with Ag-WD. CONCLUSIONS: In vitro, equine pathogens are effectively killed when exposed to Ag-WD, and the rate of bacterial killing by Ag-WD is similar to a constant silver concentration of 6.45 microg/mL. CLINICAL RELEVANCE: The in vitro antimicrobial properties of this silver-coated nylon wound dressing are promising for future prevention of equine wound infections.

A new tracheal tube for difficult intubation.

We have compared a new Portex tracheal tube with the Oxford tube in performing simulated grade 3 difficult intubations. The Portex tube was modified so that the bevel faced backwards, as in the Oxford tube. A gum elastic introducer was used with both tubes. The time taken and number of attempts needed were recorded, with changes in arterial pressure, heart rate and incidence of sore throat. Both tubes were successful in avoiding the problem of obstruction at the cords, which occurs when a standard Magill tube is used with an introducer. Thus the new tube has the merits of the Oxford tube without the disadvantages of rubber. It is suitable for both easy and difficult intubations with advantages in safety, cost and convenience. An unexpected but important finding was a clear learning effect, despite both investigators being familiar with the technique at the outset. Over the course of the study, intubation time decreased progressively (P < 0.001). This provides new evidence of the need for trainees to practise the art of intubation when the cords are not visible. Our estimate of the learning "half-life" was 15 intubations; we conclude that 30 simulated grade 3 intubations would be a reasonable objective for trainees before handling high-risk cases.

Tracheal intubation in a mannikin: comparison of the Belscope with the Macintosh laryngoscope.

Tracheal intubation carries a risk of accidental oesophageal intubation; this is increased with inexperienced trainees, and in patients with a difficult airway. The recent introduction of an angulated laryngoscope, the Belscope, may permit a better view of the vocal cords and increase the accuracy of orotracheal intubation. To determine how easy it is to learn to use the Belscope compared with the traditional Macintosh laryngoscope, a group of medical students attempted to intubate a mannikin which had been modified to simulate a difficult intubation. Time to intubation was fast with both laryngoscopes, although faster with the Macintosh, but the Belscope produced an unexpected greater incidence of failed intubation.

Pre-oxygenation: an easy method for all elective patients.

The transparent plastic facemask has been investigated as a pleasant method of pre-oxygenation for elective non-high risk cases in 60 healthy ASA I or II patients randomly allocated to two groups. The patients in the pre-oxgenated group (n = 30) received 8 litre min-1 oxygen through a plastic facemask for 3 min whereas those in a control group (n = 30) were not pre-oxygenated. In all patients anaesthesia was induced with propofol 2.5 mg kg-1, fentanyl 1 ug kg-1 and atracurium 0.6 mg kg-1. Manual ventilation of the lungs using a Mapleson A breathing system was performed for 2 min with 50% oxygen in nitrous oxide prior to oral intubation. Arterial saturation in the pre-oxygenated group rose significantly from a mean baseline value of 96.4 (+/- 0.9)% to 99 (+/- 0.8)% (P < 0.01) and then remained stable both after induction and intubation: 99.1 (+/- 0.8)% and 98.9 (+/- 1.1)% respectively. In the control group arterial saturation dropped sharply within 20 s following induction to a mean of 89.8 (+/- 3.1)%, and it was 30 s before arterial saturation reached the equivalent value in the pre-oxygenated group as a result of manual inflation of the lungs. An 8 litre min-1 oxygen flow via a standard transparent plastic facemask is a simple, feasible and acceptable method for routine pre-oxygenation for all elective cases.

Inadvertent suppression of a fixed rate ventricular pacemaker using a peripheral nerve stimulator.

Electromagnetic interference usually produces only minor effects in patients with pacemakers. Nevertheless, the possibilities of serious and even fatal consequences of this complication must be recognised. This case reports an unusual anaesthetic source of interference, caused by activation of a popular nerve stimulator, resulting in cardiac arrest in a patient with a fixed-rate ventricular pacemaker.

Endotracheal intubation skills of medical students.

The ability and confidence of clinical medical students to insert endotracheal tubes correctly and quickly and to recognize oesophageal misplacement was evaluated. Ten (33%) of the medical students intubated the trachea correctly at their first attempt but 14 (47%) incorrectly identified the position of the endotracheal tube. However, recognition improved by their second and third attempts (70% and 80% respectively). Ninety-three percent of students intubated correctly on their third attempt. Although medical students can obtain better results at correct tube placement with repeated attempts under optimum conditions--a practice effect--and do better at recognizing correct tube placement there is still a persistent failure to recognize endotracheal tube misplacement, ie oesophageal intubation. It is the ability to recognize oesophageal intubation promptly that is a life-saving skill. This essential skill should be taught during the introductory anaesthesia programme through the use of clinical patients.