Foreign gene expression in Corynebacterium pseudotuberculosis: development of a live vaccine vector.

A defined phospholipase D mutant of Corynebacterium pseudotuberculosis, designated Toxminus, was used as a live vector to express and deliver a range of candidate vaccine antigens to sheep. Expression levels of the foreign genes in Toxminus were evaluated when directed from a number of different promoters, both constitutively expressed and inducible, as fusions with expressed genes including a signal sequence, and from chromosomal and episomal loci. In general expression levels were low and it appeared that some of the recombinant proteins were tolerated by C. pseudotuberculosis Toxminus better than others. Gene expression was however sufficiently high for three of the genes to elicit antibody responses specific to the recombinant protein following a single dose of the live Toxminus vector vaccine. This work suggests that C. pseudotuberculosis Toxminus has potential for development as a live veterinary vaccine vector.

The 'easy street' myth: self harm among aboriginal and non-aboriginal female sole parents in urban state housing.

OBJECTIVE: To test the hypothesis that, controlling for socio-demographic factors, destructive behaviour among Aboriginal and non-Aboriginal female sole parents will not be significantly different. METHOD: This study took place among an urban population of sole parents in Adelaide, South Australia, living in government housing. Two sample subsets were made up of 52 Aboriginal and 45 non-Aboriginal mothers from similar postcodes. Trained interviewers administered a questionnaire which, in addition to basic demographic data, elicited information concerning finance, housing, upbringing, experience of abuse and police interaction. The major issue of concern in the study was suicide attempt. RESULTS: 1-in-3 of the whole sample, 2-in-5 of the non-Aboriginal and 1-in-4 of the Aboriginal subset had attempted suicide at least once and half more than once. Statistical differences among 'attempters' vs. 'non-attempters', irrespective of ethnicity, included increased familial alcohol abuse, physical and sexual abuse, economic difficulty, poor self esteem and perceived discriminatory treatment by welfare agencies and, in the case of Aboriginals, by police. CONCLUSION: The social environment is critical to understanding destructive behaviour, including self-harm, regardless of culture or ethnicity. The data show that suicide attempts among female sole parents in State-housing is one of the few health indices for which Aboriginal statistics are less than for non-Aboriginals. IMPLICATIONS: It is evident that class, rather than ethnicity, better explains self-harm in this urban population. It is suggested that reluctance to access services, especially in times of crisis, relates in part to perceptions of care services and that, for Aboriginals, the value of culturally appropriate community-run services have specific public health and policy implications.

LIF (AM424), a promising growth factor for the treatment of ALS.

Growth factors are theoretically promising agents for ALS therapy, but have been disappointing in subcutaneous delivery due to either toxicity or lack of major efficacy. Leukaemia inhibitory factor (LIF), was named after its effect on haemopoietic cells, and belongs to a group of cytokines which includes CNTF, IL-6, CT-1, OM and IL-11. All group members use the gp130 signal transducing subunit for intracellular signalling, but show differences in biological effect. In vitro and in vivo studies on axotomy and nerve crush models demonstrate a powerful effect of LIF in the survival of both motor and sensory neurones, while reducing denervation induced muscle atrophy. Its effects in muscle also include stimulating myoblast proliferation in vitro, and up-regulation after muscle injury. LIF will also stimulate muscle regeneration in vivo when applied exogenously after injury. In published studies of both axotomy induced neuronal death and in the Wobbler mouse models LIF is active at doses of 10 microg/kg delivered systemically, well below the expected maximum tolerated dose suggested by primate safety studies. LIF is expressed in low levels by spinal cord neurones with significant up-regulation when the neurones are damaged by BOAA toxin, an excitatory amino acid associated with a form of ALS. This augments other evidence suggesting LIF is a trauma factor playing a role in the injury response of adult neuronal tissue, and may be more effective than related growth factors. Taken together, the data suggests LIF is a physiologically relevant trophic factor with implications in clinical medicine as a therapy for ALS, and a human recombinant form (AM424), entered human clinical trials during 1998.

The impact of a computer generated patient held health record.

OBJECTIVE: To examine the use and impact of a computer generated, patient held health record (PHR) on information sharing, responsibility sharing and preventive health care. SETTING: An academic group, private solo and private group general practice in Adelaide, South Australia. METHODS: Patients with chronic health problem(s) were randomly assigned to an experimental control or post test only group. Pre and post intervention data were collected using a standardised audit and abstraction of the patient records into a computer based record system. In addition, patient and doctor questionnaires, telephone follow ups and face to face interviews were conducted. OUTCOME MEASURES: Patient and GP use of, and satisfaction with the PHR; effectiveness of information and responsibility sharing; and uptake and performance of selected preventive health care by patient and GP. RESULTS: Seventy-two patients were recruited (29 received the PHR, and 22 each were in the control and post test only groups). The PHR was well received and used in both primary and secondary care settings. No statistically significant differences in the outcome measures were found between the groups as well as before and after the intervention (Kruskal-Wallis, p > 0.05). Data trends suggested that the PHR may increase information and responsibility sharing as well as improve patient awareness of the issues involved, with patient participation in information sharing, preventive health care and clinical decision making. Provided training and resources were made available, participating GPs believed that the computer based methodology developed was a practical option for use in practice. CONCLUSION: The computer generated PHR is an important determinant of patient participation in information and responsibility sharing, health promotion, and disease management. Implementation and evaluation studies are recommended.

Immunogenicity and protection studies with recombinant mycobacteria and vaccinia vectors coexpressing the 18-kilodalton protein of Mycobacterium leprae.

The activation of antigen-specific T lymphocytes is essential for the control of leprosy infection in humans and experimental animals. T cells recognize a variety of protein antigens from Mycobacterium leprae, including the 18-kDa protein, which is limited in distribution among mycobacteria and which is absent from Mycobacterium tuberculosis and the vaccine strain, Mycobacterium bovis BCG. Adjuvant preparations of mycobacterial protein antigens have had limited protective efficacy for experimental infections in animals. Since recombinant vectors may elicit more effective T-cell responses than adjuvant preparations, recombinant vaccinia virus (VV18) and M. bovis BCG (BCG18) vectors expressing the 18-kDa protein of M. leprae were prepared. Both VV18 and BCG18 stimulated anti-18-kDa protein antibody and lymphocyte proliferative responses. Sequential immunization with VV18 followed by BCG18 induced higher levels of specific immunoglobulin G2a antibodies than immunoglobulin G1 antibodies, in contrast to immunization with VV18 or BCG18 alone. The protective efficacy of immunization with VV18 from a challenge with BCG18 was examined in two murine models of mycobacterial infection. After intravenous challenge, mice immunized with recombinant vaccinia virus exhibited lower initial levels of replication and earlier clearance of BCG18 from their spleens than mice immunized with vaccinia virus expressing an unrelated protein. After footpad infection in a dissemination model, there was earlier clearance of BCG18 from specifically immunized mice. However, immunization of mice with VV18 did not prevent a productive mycobacterial infection.

Protection of sheep against caseous lymphadenitis by use of a single oral dose of live recombinant Corynebacterium pseudotuberculosis.

An inactive form of the Corynebacterium pseudotuberculosis phospholipase D (PLD) gene was constructed and expressed in a PLD-negative strain (designated Toxminus) of C. pseudotuberculosis. Antibody responses specific to Toxminus and both Toxminus and PLD proteins were detected in sheep following oral administration of Toxminus or Toxminus expressing the PLD toxoid, respectively. However, only those sheep vaccinated with Toxminus expressing PLD toxoid were protected against wild-type challenge. These results confirm the importance of PLD as a protective antigen and demonstrate both the potential for developing an oral caseous lymphadenitis vaccine and C. pseudotuberculosis Toxminus as a live vaccine vector.

Putative functional domain within ORF2 on the Mycobacterium insertion sequences IS900 and IS902.

Repeated DNA sequences have been identified in a range of mycobacterial species and have been implicated in the increased virulence of some of these species, namely, Mycobacterium paratuberculosis and M. avium subsp. silvaticum. Here we present a case to suggest that the insertion sequences IS900 and IS902 encode a protein from a putative gene positioned on the complementary strand to their transposase genes. Based on amino acid homology analyses, this open reading frame (ORF2) could encode a transport protein. The ORF2 protein thus IS900 and IS902, may have a role in the increased pathogenicity of M. paratuberculosis and M. avium subsp. silvaticum from an M. avium background.

Expression of ovine gamma interferon in Escherichia coli and Corynebacterium glutamicum.

Bacteria of two species, Escherichia coli and Corynebacterium glutamicum, were used as hosts to express recombinant ovine gamma interferon as a fusion protein with glutathione S-transferase. The recombinant gamma interferon produced by both bacteria was biologically active in vitro and was recognized by anti-gamma interferon monoclonal antibodies. E. coli produced large amounts of soluble recombinant protein which could be purified by a simple affinity chromatography method. Only a small fraction of the recombinant protein made by C. glutamicum was recovered by this method. Expression of recombinant protein in C. glutamicum was unstable but could be controlled by increased regulation of the tac promoter. Both hosts expressed ovine gamma interferon at high levels, with the recombinant protein making up a significant proportion of the cellular protein content.

Diagnosis and epidemiology of bovine tuberculosis using molecular biological approaches.

Conventional methods for diagnosis and epidemiological studies of bovine tuberculosis and other mycobacterial diseases are far from ideal. In the last twelve years, molecular biology has provided new approaches which have enabled detailed studies to be made of the molecular characteristics of Mycobacterium bovis, the causative organism of bovine tuberculosis. These characteristics have been investigated for their potential use in diagnosis and epidemiological studies. Restriction fragment analysis of genomic DNA from isolates of M. bovis has provided a highly discriminatory typing system which has been used extensively for epidemiological studies. DNA elements in M. bovis have been investigated for their potential use in diagnostic assays based on the polymerase chain reaction. Individual antigens of M. bovis and their reactive epitopes have been identified and investigated for their usefulness in both serological and T cell assays of animal infection. Serological assays have proven disappointing and the full potential of T cell assays has yet to be determined. Currently, T cell assays based on recombinant antigens have not been as sensitive as those based on natural antigens.

A pattern of decline in general practice obstetric care in Adelaide.

Some educational, training and politico-economic reasons for the decline in GP obstetrics in Adelaide, South Australia, are described. It is recommended that similar studies be done in other centres in Australia to evaluate the general nature of the findings.

A nursing attachment for medical students at Flinders University.

OBJECTIVE: To determine medical student response to the set objectives for a one-week nursing attachment, completed before their major clinical training, in a teaching hospital. DESIGN: Linear ordinal scale assessments of the objectives, encompassing familiarization with ward and hospital practice, appreciation of the roles of nurses in patient care, the interaction between nursing and medical professions, and the acquisition of basic skills related to patient care and focus group discussions with students and with their nurse supervisors. PARTICIPANTS: One hundred and sixty-four medical students at Flinders University who completed their nursing attachments in 1986 and 1990-1992. RESULTS: Students thought there had been a uniformly high level of achievement of objectives. A number of suggestions were made by both students and supervisors to improve the program. CONCLUSIONS: A period of nursing attachment for medical students, before their major clinical training, helps them to fit into usual ward routine, provides them with some basic patient care skills, and helps them gain greater understanding of and respect for the roles and responsibilities of nurses.

Characterisation of a highly repeated DNA sequence from Mycobacterium bovis.

We report characterisation of a novel repeat sequence from a Mycobacterium bovis genomic library. The highly repeated sequence belongs to a family consisting of a 24 base pair (bp) direct repeat (DR), that appears to be organized into clusters on the chromosome. We classify the 24-bp DR into the group of prokaryotic DNA repeats known as the interspersed repetitive sequence elements. The 24-bp DR will be of potential use as a DNA fingerprinting tool in epidemiological studies of M. bovis.

Removal of false-positive reactions from plasma in an enzyme immunoassay for bovine interferon-gamma.

A monoclonal antibody-based sandwich enzyme immunoassay (EIA) for bovine interferon-gamma (IFN-gamma) has been developed and can be used in conjunction with a whole blood culture system to diagnose tuberculosis in cattle. During its development, normal bovine plasma samples were tested to establish background levels of circulatory IFN-gamma. Of 191 samples tested, 81 (42.4%) were positive (OD > 0.1) when tested undiluted in intact monoclonal antibody (IgG1)-coated wells compared to only 8 (4.2%) in F(ab')2-coated wells, which suggested non-specific interference in the EIA rather than circulatory IFN-gamma. Reactivity of all remaining samples was removed by diluting plasmas 1/2 with 1% casein-PBS-0.05% Tween 20 supplemented with an optimum amount (5%) of normal mouse serum (NMS). Serum pools derived from BALB/c, DBA/2, C3H/HeJ, CBA/CaH and Swiss, but not C57BL/6J, mice were found to inhibit equally the reactions of five strong false-positive bovine plasma samples but had no effect on the titre of IFN-gamma in the sample. Sera from other species tested were less effective. This suggests that the interfering factors possess a high degree of specificity, since the immunoglobulin heavy chain of IgG1 produced by all these five strains of mice are allotypically identical and different to IgG1 produced by C57BL/6J mice. The use of F(ab')2 antibody fragments to coat plate wells and sample diluent containing 5% NMS has resulted in an EIA for bovine IFN-gamma that is virtually free from false-positive reactions, has a high degree of reproducibility and a sample detection limit equivalent to approximately 80 pg/ml recombinant bovine IFN-gamma.

Characterisation of a novel repetitive DNA sequence from Mycobacterium bovis.

We report characterisation of three copies of a novel repeat sequence isolated from a Mycobacterium bovis genomic library. The repeat occurs within open reading frames, potentially encoding a conserved tandem array of a pentapeptide sequence with the consensus X-Gly-Asn-X-Gly. The tandem array is present up to five times in M. bovis and it is proposed that they may occur in a family of genes expressing functionally related proteins. We postulate that these proteins may play a role in binding of M. bovis to host cell receptors.

Rational attenuation of Corynebacterium pseudotuberculosis: potential cheesy gland vaccine and live delivery vehicle.

The phospholipase D (PLD) gene (pld) has been deleted from the Corynebacterium pseudotuberculosis chromosome by using site-specific mutagenesis. Sheep infection trials indicate that the PLD-negative C. pseudotuberculosis strain (Toxminus) is incapable of inducing caseous lymphadentis (cheesy gland) even at doses two logs higher than that at which the wild-type strain produces the disease. This clearly establishes PLD as a major C. pseudotuberculosis virulence factor. Vaccination of sheep with live Toxminus C. pseudotuberculosis elicits strong humoral and cell-mediated immune responses and protects the animals from wild-type challenge.

A randomised study of outcomes in a defined group of acutely ill elderly patients managed in a geriatric assessment unit or a general medical unit.

The aim of this study was to identify differences in the medical management and clinical outcome in a group of elderly patients admitted to a designated geriatric assessment unit (GAU) or to two general medical units (GMUs). A prospective randomised controlled trial was undertaken in 267 patients aged 70 years and over (mean age = 78.3 years). Following discharge from hospital, patients were followed up at three monthly intervals for a total of 12 months. At the time of discharge, no significant differences were found in inpatient management, length of stay, mortality rates, discharge rates to institutional care or utilisation of community services in patients admitted to the GAU and the GMUs. Similarly, no significant differences were found at three, six, nine, and 12 month follow up in case fatality, activities of daily living indices, mental health status, rates of institutional referral and the level of community service support in patients admitted to the GAU and the GMUs studied. These findings do not show any advantage for the unselected 70+ acutely ill elderly patient who is admitted to a designated geriatric assessment unit rather than to a general medical unit. Therefore, an admission policy to GAU, based solely on age 70+ is medically inappropriate and cost-inefficient. Evidence from other sources suggests that an age cohort of acutely admitted patients beyond 80 years may well have returned more optimistic findings for the GAU. In future, GAUs will require a more selective admission policy to maximise the benefits of their rehabilitative and interdisciplinary approach.