Risk of Mycoplasma bovis transmission from contaminated sand bedding to naive dairy calves.

The objective of this study was to evaluate the possible transmission of Mycoplasma bovis from positive sand bedding to naïve dairy calves. Twelve preweaned Holstein bull calves were blocked in pairs and randomly assigned as unexposed controls (n=6) bedded with control sand, or exposed calves (n=6) bedded with sand previously positive for M. bovis at a dairy farm. Bedding sand was cultured weekly. Nasal and ear swabs and sera were collected weekly, tracheal swabs were collected monthly, and by the end of the 105-d study, all calves were euthanized (n=10) or died (n=2). Sera were tested for M. bovis-specific antibody. Mycoplasma spp. culture was performed on nasal and ear swabs; culture and a PCR differentiating multiple Mycoplasma spp. were performed on postmortem samples of lung, retropharyngeal lymph node, and trachea from each calf. A complete necropsy also was performed. During 6 wk, mycoplasma concentration in exposed group sand was between 200 and 32,000 cfu/g. All 166 tracheal swabs, nasal and ear swabs, and postmortem tests from all calves were negative for mycoplasma. All 94 sera were negative for M. bovis-specific antibody. No gross pathology suggestive of mycoplasma disease was detected. The probability of mycoplasma detection, if an exposed calf had become infected 4 wk after exposure, ranged between 97 and 99% depending on time of exposure for individual calves. There was no evidence that sand bedding contaminated with M. bovis might serve as a source of transmission to naïve dairy calves.

The Mental Capacity Act 2005--implications for anaesthesia and critical care.

The Mental Capacity Act 2005 is due to come into force in April 2007. The Act provides a protective statutory framework for decision-making on behalf of incompetent adults, representing, in the main, a codification of the common law that had already developed in this area. For example, 'advance decisions' are now given formal statutory recognition. Importantly, the Act creates a new specialist 'Court of Protection' to manage the Act's enforcement, and an office of 'Public Guardian' to act as registering authority for new 'Lasting Powers of Attorney' and 'court-appointed deputies', both of which will be able to make proxy decisions about medical treatment for adult patients without capacity. There is also considerable regulation concerning the participation of adults without capacity in research. Given that their practice routinely involves the medical treatment of adults who lack legal capacity, anaesthetists and intensivists should familiarise themselves with the Act's key precepts.

Inhibition of influenza virus infections in immunosuppressed mice with orally administered peramivir (BCX-1812).

Experiments were run to determine the effect of oral gavage treatment with the cyclopentane influenza virus neuraminidase inhibitor peramivir (BCX-1812, RWJ-270201) in influenza A (H1N1) virus-infected mice that had their immune system suppressed by cyclophosphamide (CP) therapy or in severe combined immune deficient (SCID) mice. Treatment of CP-immunosuppressed mice with peramivir using doses of 100, 10, or 1mg/kg/day was begun 2.5 or 8 days post-virus exposure and continued twice daily for 3 or 5 days. The 5-day therapy was more effective than the 3-day treatment, as seen by significantly increased survivor numbers, lessened decline in arterial oxygen saturation, reduced lung consolidation, and inhibition of lung virus titers. Infected SCID mice were also responsive to peramivir therapy begun 8 days after virus exposure and continued for 5 days, although antiviral effects did not include prevention of death and were dependent upon the viral challenge dose received. These data indicate that peramivir may have potential for treatment of influenza virus-infected immunosuppressed patients.

Consent for anaesthesia.

Current professional guidelines concerning information and consent for anaesthesia are a fair representation of English law. However, they reject the need for specific, written consent for anaesthesia, a position which is in accordance with other Western jurisdictions. This is understandable, as there would be a number of problems inherent in such an approach: the consent process would be unnecessarily labour and time intensive, the generic nature of the information to be disclosed would not allow for operator-dependent variables, and many of the disclosable risks continue to be of uncertain incidence. Moreover, written consent is not needed in order to defend cases of assault by anaesthetists. However, for the very reason that there are a large number of risks associated with anaesthesia (risks that are unknown to the majority of surgeons), together with the possibility of the courts moving towards a reasonable patient standard of information disclosure (as a result of the introduction of human rights legislation into English law), it is our view that the Association of Anaesthetists of Great Britain and Ireland should change their guidelines and advise anaesthetists to obtain separate, written affirmation from patients that certain risks and consequences of anaesthesia have been explained to them. In addition, a standardised consent form for anaesthesia may prove invaluable in retrospectively defending a claim of negligence founded around information disclosure, by recording exactly the risks and consequences of interventions discussed by the anaesthetist and the patient.

The Human Rights Act 1998: implications for anaesthesia and intensive care.

The Human Rights Act 1998 was incorporated into UK statutory law on October 2, 2000. The 18 Articles of the Act are likely to have a significant impact on the practice of medicine in the UK, particularly in reference to consent, disclosure of medical information and patient access to healthcare. This article examines the implications of the new legislation for anaesthetic and intensive care practice.

Myxobolus cerebralis infection in rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) exposed under natural stream conditions.

From early April into mid-June 1977, sequential groups of juvenile rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) were each exposed for 10 days to the parasite Myxobolus cerebralis by immersion in a stream inhabited by infected wild trout. Following incubation in a M. cerebralis-free facility, trout were subsequently killed, and heads and gill arches were examined by routine histologic methods. A grading scale to quantify lesion severity was developed and applied. Percentage infected, lesion severity scores, effects of water temperature and flow rates on percentage infected and lesion severity scores, and resulting pathology were determined for each species at each exposure period. The percentage of rainbow trout infected with M. cerebralis was significantly higher than the percentage of brown trout infected for each exposure period. The percentages of rainbow trout infected in exposure periods later in the calendar year were significantly higher than those in earlier periods. The percentages of brown trout infected were not significantly different among exposure periods. Overall average lesion severity scores were significantly higher in rainbow than in brown trout. Lesion severity scores in rainbow trout increased over time (a positive correlation with exposure period). Lesion severity scores were not significantly different for brown trout among exposure periods. A significant correlation existed between water temperature and percentage of rainbow trout infected; a significant correlation also existed between water temperature and lesion severity scores in rainbow trout. Similar correlations did not exist for percentage of brown trout infected or accompanying lesion severity scores. In rainbow trout, ventral calvarium was the most common site of M. cerebralis replication, followed by gill arches. In brown trout, lesions were virtually confined to gill arches. Early lesions consisted of foci of cartilage necrosis with small numbers of M. cerebralis developmental stages. More advanced lesions consisted of multifocal areas of cartilage necrosis with numerous M. cerebralis developmental stages and/or mature myxospores bordered and/or infiltrated by mono- and multinuclear leukocytes. Lesions in brown trout were smaller and had fewer associated leukocytes and M. cerebralis developmental stages and/or mature myxospores. Higher infection rates, lesion severity scores, and differences in lesion location in rainbow versus brown trout explain in part why numbers of rainbow but not brown trout have fallen in western rivers inhabited with M. cerebralis-infected trout.

Coinfection with influenza B virus does not affect association of Neisseria meningitidis with human nasopharyngeal mucosa in organ culture.

There is an epidemiological association between influenza virus infection and meningococcal disease. Proposed mechanisms are the destruction of the normal epithelial barrier function of the upper respiratory tract by influenza virus or the expression of human or viral surface-exposed proteins that enhance bacterial adherence and/or invasion. To test these hypotheses, human nasopharyngeal mucosa specimens from a total of 19 individual donors were successfully infected with influenza B virus and then inoculated with serogroup B Neisseria meningitidis. Subsequent bacterial association with the epithelial surface was measured in three separate series of experiments by using transmission electron microscopy (n = 6), scanning electron microscopy (n = 6), and counting of viable bacteria within homogenates of explants (n = 7). Penetration of the mucosa was estimated by measuring the count of viable bacteria recovered from explants after exposure to sodium taurocholate. Bacterial association with the surface of explants was time dependent over 24 h of superinfection. Influenza virus did not positively or negatively influence bacterial attachment to or penetration of explant mucosa compared to those of uninfected controls, even when the period of preincubation with virus was extended to 7 days. When proteins were purified from mucosal epithelium and immobilized on nitrocellulose membranes, N. meningitidis attached predominantly to bands corresponding to proteins of 210 and 130 kDa. In the presence of influenza virus infection, these proteins were gradually lost over the course of 72 h. In conclusion, influenza B virus did not increase association of serogroup B N. meningitidis with human nasopharyngeal mucosa.

Heterogeneity in responses by primary adult human colonic epithelial cells to purified enterotoxin of Bacteroides fragilis.

BACKGROUND: Enterotoxigenic strains of Bacteroides fragilis (ETBF) have been implicated in diarrhoeal illness in livestock and children, but their role in adult human colonic disease is unknown. AIMS: To investigate responses by primary adult human colonic epithelial cells to purified B fragilis toxin (BFT). METHODS: BFT was purified from culture supernatant of a highly toxigenic strain of ETBF. Morphological changes to primary colonic epithelial cells, in response to purified BFT, were studied in organ culture of colonic biopsy specimens from 15 adults. RESULTS: BFT induced epithelial cell cytotoxicity in colonic biopsy specimens from 12/15 subjects. The BFT induced morphological changes were characterised by epithelial cell rounding, separation from adjacent cells, and detachment from the basement membrane. In severely affected specimens, almost all the epithelial cells were affected. There was heterogeneity between subjects in the rate at which BFT induced epithelial cell cytotoxicity occurred. Furthermore, in colonic biopsy specimens from three subjects, exposure to BFT did not induce any significant morphological changes to epithelial cells. CONCLUSION: BFT is capable of inducing cytotoxicity in primary adult human colonic epithelial cells. Such an effect of ETBF derived BFT on epithelial cells in the colon in vivo would be expected to lead to mucosal inflammation and diarrhoea. Heterogeneity in responses by primary colonocytes probably reflects the outcome of host-BFT interactions. Such interactions in vivo could determine the occurrence of colonic disease in some individuals but not others.

The 18th C.L. Oakley Lecture. Pathogenicity of enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) remain an important world-wide cause of diarrhoeal disease and mortality of infants and young children. Research programmes around the world have, in recent times, made enormous strides towards a better understanding of EPEC pathogenesis, yielding unique insights into the molecular intercourse between host and pathogen. Recombinant DNA and cell biology techniques have provided powerful tools, giving the first intriguing glimpses of a wealth of bacterial products mediating complex host:pathogen interactions involving the subversion of normal host signalling processes. Much has been discovered since 1945, when E. coli was first implicated as a cause of diarrhoea. However, many questions remain unanswered and many more remain unasked. Much remains to be discovered, especially in the area of molecular interactions between host and pathogen and how they relate to the manifestation of disease in the patient.

Cloning, sequencing, characterisation and implications for vaccine design of the novel dihydrolipoyl acetyltransferase of Neisseria meningitidis.

A lambdaZap-II expression library of Neisseria meningitidis was screened with a rabbit polyclonal antiserum (R-70) raised against c. 70-kDa proteins purified from outer membrane vesicles by elution from preparative SDS-polyacrylamide gels. Selected clones were isolated, further purified, and their recombinant pBluescript SKII plasmids were excised. The cloned DNA insert was sequenced from positive clones and analysed. Four open reading frames (ORFs) were identified, three of which showed a high degree of homology with the pyruvate dehydrogenase (E1p), dihydrolipoyl acetyltransferase (E2p) and dihydrolipoyl dehydrogenase (E3) components of the pyruvate dehydrogenase complex (PDHC) of a number of prokaryotic and eukaryotic species. Sequence analysis indicated that the meningococcal E2p (Men-E2p) contains two N-terminal lipoyl domains, an E1/E3 binding domain and a catalytic domain. The domains are separated by hinge regions rich in alanine, proline and charged residues. Another lipoyl domain with high sequence similarity to the Men-E2p lipoyl domain was found at the N-terminal of the E3 component. A further ORF, coding for a 16.5-kDa protein, was found between the ORFs encoding the E2p and E3 components. The identity and functional characteristics of the expressed and purified heterologous Men-E2p were confirmed as dihydrolipoyl acetyltransferase by immunological and biochemical assays. N-terminal amino-acid analysis confirmed the sequence of the DNA-derived mature protein. Purified Men-E2p reacted with monospecific antisera raised against the whole E2p molecule and against the lipoyl domain of the Azotobacter vinelandii E2p. Conversely, rabbit antiserum raised against Men-E2p reacted with protein extracts of A. vinelandii, Escherichia coli and N. gonorrhoeae and with the lipoyl and catalytic domains of E2p obtained by limited proteolysis. In contrast, the original R-70 antiserum reacted almost exclusively with the lipoyl domain, indicating the strong immunogenicity of this domain. Antibodies to Men-E2p were detected in patients and animals (rabbits and mice) infected with homologous or heterologous meningococci or other neisserial species. These results have important implications for the understanding of PDHC and the design of future outer membrane vesicle-based vaccines.

Acceptance of skin grafts by isogenic rainbow trout (Oncorhynchus mykiss).

OBJECTIVE: To test the immunocompetence of isogenic families of rainbow trout by measuring their ability to accept or reject skin grafts. ANIMALS: 3 families of isogenic rainbow trout (Oncorhynchus mykiss), produced by mating homozygous females and homozygous males, plus 4 chinook salmon (O tshawytscha) were used in these experiments. PROCEDURE: Grafts (allografts, members of the same family; autografts, donor and recipient were the same fish; and xenografts, O tshawytscha as donor) were exchanged. Grafts were applied on day 0 and removed on day 21, placed in neutral-buffered formalin, and embedded in paraffin. Lymphocytes and nuclei were counted in representative stained sections in the epidermis, dermis, and hypodermis. Results were analyzed by univariate analysis, using the Shapiro-Wilk statistic. RESULTS: Autografts were retained and minimal histologic changes were apparent. Allografts were histologically similar to autografts. Xenografts were rejected. CONCLUSIONS: Results indicate that the immune system of isogenic rainbow trout is unable to distinguish between family members within isogenic families, but that a vigorous response is mounted against chinook salmon xenografts. The isogenic rainbow trout are immunocompetent with respect to the phenomenon of graft rejection.

Phosphorylation of myosin light chain at distinct sites and its association with the cytoskeleton during enteropathogenic Escherichia coli infection.

Myosin light chain, the most prominent host cell phosphoprotein during adhesion of enteropathogenic Escherichia coli to cultured HEp-2 cells, was shown to be distributed between cytosolic and cytoskeletal cell fractions; its association with the cytoskeletal fraction increased with time of enteropathogenic E. coli incubation. Phosphopeptide mapping indicated cytosolic and cytoskeletal myosin light chain phosphorylation at different sites by protein kinase C and myosin light chain kinase.

Elucidation of the molecular actions of NCAM and structurally related cell adhesion molecules.

The Neural Cell Adhesion Molecule (NCAM) is a founder member of a large family of cell surface glycoproteins that share structural motifs related to immunoglobulin and fibronectin type III (FN III) domains [Walsh and Doherty (1991) (Fig. 1). These glycoproteins have been grouped based on the respective number of each type of domain. In vertebrates members of this family of glycoproteins include L1/NILE, NgCAM, axonin-1/TAG-1, and Thy-1 as well as NCAM. In addition structural homologs of NCAM and L1 have been identified in Drosophila and Grasshoppers [Walsh and Doherty (1991)]. These insect homologs are called fasciclins and a series of mutants corresponding to these aspects of synaptic plasticity [Mayford et al. (1992) Science 256:638-644]. In vertebrates all of these glycoproteins are expressed in the developing nervous system where they have been identified as candidate molecules for mediating axon outgrowth, fasciculation, regeneration, and target recognition. In addition, NCAM is expressed in a number of different tissues and cell types. For example, NCAM is expressed in a dynamic pattern in developing and regenerating adult muscle. In this review we aim to describe important aspects of the role of these CAMS in development of the nervous system, including the neuromuscular junction. Furthermore, we will explore the prospective use of molecular biology, cell biology, and molecular genetic techniques, such as transgenic mice, to understand the role and molecular action of this family of cell adhesion molecules in vivo.

Experimental infections of Sarcocystis spp. in Rocky Mountain elk (Cervus elaphus) calves.

Four 4-mo-old elk calves (Cervus elaphus) obtained from northeastern Oregon (USA) each were inoculated orally with 250,000 sporocysts of Sarcocystis spp., including S. sybillensis and S. wapiti. Three similar elk calves of comparable ages and weights served as uninoculated controls maintained with the inoculated elk during the experimental period between September and December 1993. Body weights were evaluated at 0 and 90 days postinoculation (PI); packed cell volumes of whole blood were evaluated at 0, 30, and 60 days PI, and numbers of sarcocysts in histologic sections from 11 selected tissues were evaluated at 90 days PI. Significant differences in blood packed cell volumes were not detected between groups (P > 0.05). Except for weight gain, elk remained healthy. Mean (+/- SE) weight gain of inoculated elk (27.1 +/- 1.6 kg) was significantly (P < 0.05) less than that of controls (40.2 +/- 4.9 kg). Mean (+/- SE) number of sarcocysts in tissues of inoculated (114.4 +/- 25.7 cm2) and controls (4.5 +/- 1.4 cm2) differed significantly (P < 0.05). Heart, esophagus and skeletal muscle contained the most sarcocysts. No sarcocysts were detected in brain, spinal cord, or testicles. Histologically, mononuclear myositis and myocarditis, with numerous intralesional sarcocysts were seen. Less severe, but widespread inflammation occurred in brain, spinal cord, and optic nerve. Mortality and anemia were not seen, but weight gain depression was detected in the inoculated elk over the 90 day experimental period.

Primary structure and functional expression of a mouse inward rectifier potassium channel.

A complementary DNA encoding an inward rectifier K+ channel (IRK1) was isolated from a mouse macrophage cell line by expression cloning. This channel conducts inward K+ current below the K+ equilibrium potential but passes little outward K+ current. The IRK1 channel contains only two putative transmembrane segments per subunit and corresponds to the inner core structure of voltage-gated K+ channels. The IRK1 channel and an ATP-regulated K+ channel show extensive sequence similarity and constitute a new superfamily.