Owner reported diseases of working equids in central Ethiopia.

REASONS FOR PERFORMING STUDY: Working horses, donkeys and mules suffer from numerous diseases and clinical problems. However, there is little information on what owners perceive as important health concerns in their working animals. OBJECTIVES: To identify and prioritise with owners the diseases and other health concerns in working equids in central Ethiopia using participatory methodologies. STUDY DESIGN: Participatory situation analysis (PSA). METHODS: The study was conducted with carthorse- and donkey-owners in 16 sites in central Ethiopia. Multiple participatory methodologies were utilised, including ranking, matrices and focus group discussions. Owners' perceptions on frequency, importance, morbidity and mortality of volunteered diseases and the clinical signs that owners attributed to each disease were obtained; information regarding the impact of these diseases and health concerns was also sought. RESULTS: A total of 40 separate disease and health problems were volunteered by carthorse- and donkey-owners. Horse-owners volunteered a musculoskeletal syndrome (with the local name 'bird', clinical signs suggest possible disease pathologies including equine exertional rhabdomyolysis), colic and epizootic lymphangitis most frequently, whereas donkey-owners volunteered sarcoids, nasal discharge and wounds to occur most frequently. One problem (coughing) was volunteered frequently by both horse- and donkey-owners. Owners demonstrated knowledge of differing manifestations and severity of these problems, which resulted in differing impacts on the working ability of the animal. CONCLUSIONS: Although many of the diseases and clinical signs had been previously reported, this study also identified some previously unreported priorities such as rabies in donkeys, an unidentified musculoskeletal syndrome in horses and respiratory signs in both horses and donkeys. The information gathered during this participatory study with owners may be used to inform future veterinary and educational programme interventions, as well as identify future research priorities.

A cluster-randomised controlled trial to compare the effectiveness of different knowledge-transfer interventions for rural working equid users in Ethiopia.

There have been few studies evaluating the efficacy of knowledge-transfer methods for livestock owners in developing countries, and to the authors' knowledge no published work is available that evaluates the effect of knowledge-transfer interventions on the education of working equid users. A cluster-randomised controlled trial (c-RCT) was used to evaluate and compare the effectiveness of three knowledge-transfer interventions on knowledge-change about equid health amongst rural Ethiopian working equid users. Groups were exposed to either; an audio programme, a village meeting or a diagrammatic handout, all of which addressed identical learning objectives, and were compared to a control group which received no intervention. Thirty-two villages were randomly selected and interventions randomly assigned. All participants in a village received the same intervention. Knowledge levels were assessed by questionnaire administration. Data analysis included comparison of baseline data between intervention groups followed by multilevel linear regression models (allowing for clustering of individuals within village) to evaluate the change in knowledge between the different knowledge-transfer interventions. A total of 516 randomly selected participants completed the pre-intervention questionnaire, 504 of whom undertook the post-dissemination questionnaire, a follow up response rate of 98%. All interventions significantly improved the overall 'change in knowledge' score on the questionnaire compared to the control, with the diagrammatic handout (coefficient (coef) 9.5, S.E.=0.6) and the village meeting (coef 9.7, S.E.=0.6) having a significantly greater impact than the audio programme (coef 4.8, S.E.=0.6). Covariates that were different at baseline, and which were also significant in the final model, were age and pre-intervention score. Although they had a minimal effect on the intervention coefficients there was a significant interaction between age and intervention. This study should aid the design of education materials for adult learning for working equid users and other groups in developing countries.

Evaluation of suicide awareness programmes delivered to veterinary undergraduates and academic staff.

In an effort to increase suicide awareness skills among veterinary undergraduates, a three-hour suicide awareness workshop (safeTALK) was delivered to third-year Royal (Dick) School of Veterinary Studies undergraduates as part of their professional development curriculum. Students were able to opt out of the session by contacting the course organisers. A total of 26 of 151 (17 per cent) third-year students attended the workshop, and 17 completed a feedback questionnaire. The vast majority of the students reported that after completing the workshop they were more likely or much more likely to recognise the signs of a person at risk of suicide, approach a person at risk of suicide, ask a person about suicide, and connect a person at risk of suicide with help. Five veterinary academics attended a two-day Applied Suicide Intervention Skills Training (ASIST) course, and all reported that the course was effective in improving suicide awareness and intervention skills.

Clinical and subclinical mastitis in smallholder dairy farms in Tanzania: risk, intervention and knowledge transfer.

In a cross-sectional study of 400 randomly selected smallholder dairy farms in the Tanga and Iringa regions of Tanzania, 14.2% (95% confidence interval (CI)=11.6-17.3) of cows had developed clinical mastitis during the previous year. The point prevalence of subclinical mastitis, defined as a quarter positive by the California Mastitis Test (CMT) or by bacteriological culture, was 46.2% (95% CI=43.6-48.8) and 24.3% (95% CI=22.2-26.6), respectively. In a longitudinal disease study in Iringa, the incidence of clinical mastitis was 31.7 cases per 100 cow-years. A randomised intervention trial indicated that intramammary antibiotics significantly reduced the proportion of bacteriologically positive quarters in the short-term (14 days post-infusion) but teat dipping had no detectable effect on bacteriological infection and CMT positive quarters. Other risk and protective factors were identified from both the cross-sectional and longitudinal included animals with Boran breeding (odds ratio (OR)=3.40, 95% CI=1.00-11.57, P<0.05 for clinical mastitis, and OR=3.51, 95% CI=1.29-9.55, P<0.01 for a CMT positive quarter), while the practice of residual calf suckling was protective for a bacteriologically positive quarter (OR=0.63, 95% CI=0.48-0.81, P

The effects of different knowledge-dissemination interventions on the mastitis knowledge of Tanzanian smallholder dairy farmers.

We developed three different knowledge-dissemination methods for educating Tanzanian smallholder farmers about mastitis in their dairy cattle. The effectiveness of these methods (and their combinations) was evaluated and quantified using a randomised controlled trial and multilevel statistical modelling. To our knowledge, this is the first study that has used such techniques to evaluate the effectiveness of different knowledge-dissemination interventions for adult learning in developing countries. Five different combinations of knowledge-dissemination method were compared: 'diagrammatic handout' ('HO'), 'village meeting' ('VM'), 'village meeting and video' ('VM+V'), 'village meeting and diagrammatic handout' ('VM+HO') and 'village meeting, video and diagrammatic handout' ('VM+V+HO'). Smallholder dairy farmers were exposed to only one of these interventions, and the effectiveness of each was compared to a control ('C') group, who received no intervention. The mastitis knowledge of each farmer (n=256) was evaluated by questionnaire both pre- and post-dissemination. Generalised linear mixed models were used to evaluate the effectiveness of the different interventions. The outcome variable considered was the probability of volunteering correct responses to mastitis questions post-dissemination, with 'village' and 'farmer' considered as random effects in the model. Results showed that all five interventions, 'HO' (odds ratio (OR)=3.50, 95% confidence intervals (CI)=3.10, 3.96), 'VM+V+HO' (OR=3.34, 95% CI=2.94, 3.78), 'VM+HO' (OR=3.28, 95% CI=2.90, 3.71), 'VM+V' (OR=3.22, 95% CI=2.84, 3.64) and 'VM' (OR=2.61, 95% CI=2.31, 2.95), were significantly (p<0.0001) more effective at disseminating mastitis knowledge than no intervention. In addition, the 'VM' method was less effective at disseminating mastitis knowledge than other interventions. Combinations of methods showed no advantage over the diagrammatic handout alone. Other explanatory variables with significant positive associations on mastitis knowledge included education to secondary school level or higher, and having previously learned about mastitis by reading pamphlets or attendance at an animal-health course.

Side-effects of antituberculosis drug treatment in patients with chronic renal failure.

Patients with chronic renal failure (CRF) have a high incidence of tuberculosis (TB). Those from the Indian subcontinent are at particular risk. The frequency of side-effects associated with antituberculous treatment in a group of patients with CRF was studied. All cases of TB in patients with CRF occurring over a 13-yr period at the Manchester Royal Infirmary, from 1986-1999, were identified by diagnostic coding, microbiology records and a TB database. The case notes were then reviewed. Twenty-four cases were identified, eight predialysis and 16 requiring regular dialysis. TB occurring in the dialysis group was extrapulmonary in every case. Nineteen of 24 (79%) patients were of Indian subcontinent origin and 14 of 16 (87%) dialysis patients were non-Caucasian. Adverse effects of treatment occurred in two of eight (25%) in the predialysis group and nine of 16 (56%) of the dialysis group. These were most commonly neuropsychiatric (6), hepatic (4) and gastrointestinal (4). Neuropsychiatric symptoms occurred exclusively in dialysis patients. In conclusion, a high incidence of side-effects from antituberculous medication, especially neuropsychiatric, hepatic and gastrointestinal, was identified in patients with chronic renal failure. Careful monitoring for side-effects is essential in this group, and consideration should be given to administering antituberculous chemoprophylaxis to all high-risk groups.

Crystal structure of the lambda repressor C-terminal domain octamer.

The three-dimensional structure of the lambda repressor C-terminal domain (CTD) has been determined at atomic resolution. In the crystal, the CTD forms a 2-fold symmetric tetramer that mediates cooperative binding of two repressor dimers to pairs of operator sites. Based upon this structure, a model was proposed for the structure of an octameric repressor that forms both in the presence and absence of DNA. Here, we have determined the structure of the lambda repressor CTD in three new crystal forms, under a wide variety of conditions. All crystals have essentially the same tetramer, confirming the results of the earlier study. One crystal form has two tetramers bound to form an octamer, which has the same overall architecture as the previously proposed model. An unexpected feature of the octamer in the crystal structure is a unique interaction at the tetramer-tetramer interface, formed by residues Gln209, Tyr210 and Pro211, which contact symmetry-equivalent residues from other subunits of the octamer. Interestingly, these residues are also located at the dimer-dimer interface, where the specific interactions are different. The structures thus indicate specific amino acid residues that, at least in principle, when altered could result in repressors that form tetramers but not octamers.

Crystallographic analysis of Lac repressor bound to natural operator O1.

Previous structures of Lac repressor bound to DNA used a fully symmetric "ideal" operator sequence that is missing the central G-C base-pair present in the three natural operator sequences. Here we have determined the X-ray crystal structure of a dimeric Lac repressor bound to a 22 base-pair DNA with the natural operator O1 sequence and the anti-inducer ONPF, at 4.0 A resolution. The natural operator is bent in the same way as the symmetric sequence, due to the binding of the hinge helices of the repressor to the minor groove at the central GCGG sequence of O1. Comparison of the structures of the repressor bound to the natural and symmetric operators shows very similar overall structures, with only slight rearrangements of the headpiece domains of the repressor. Analysis of crystals with iodinated DNA shows that the operator is uniquely positioned and allows for the sequence registration of the DNA relative to the repressor to be determined. The kink in the operator is centered between the left half-site and the central G-C base-pair of O1. Our results are most consistent with a previously proposed model in which, relative to the complex with the symmetric operator, the repressor accommodates binding to the natural operator sequence by shifting the position of the right headpiece by one base-pair step towards the center of O1.

Structure of a variant of lac repressor with increased thermostability and decreased affinity for operator.

A single amino acid substitution, K84L, in the Escherichia coli lac repressor produces a protein that has substantially increased stability compared to wild-type. However, despite the increased stability, this altered tetrameric repressor has a tenfold reduced affinity for operator and greatly decreased rate-constants of inducer binding as well as a reduced phenotypic response to inducer in vivo. To understand the dramatic increase in stability and altered functional properties, we have determined the X-ray crystal structures of a dimeric repressor with and without the K84L substitution at resolutions of 1.7 and 3.0 A, respectively. In the wild-type dimer, K84-11, Lys84 forms electrostatic interactions at the monomer-monomer interface and is partially exposed to solvent. In the K84L-11 substituted protein there is reorientation of the N-subdomains, which allows the leucine to become deeply buried at the monomer-monomer interface. This reorientation of the N-subdomains, in turn, results in an alteration of hydrogen bonding, ion pairing, and van der Waals interactions at the monomer-monomer interface. The lysine residue at position 84 appears to exert its key effects by destabilizing the "optimal" conformation of the repressor, effectively loosening the dimer interface and allowing the repressor to adopt the conformations necessary to function as a molecular switch.

Peritoneal tuberculosis in patients receiving continuous ambulatory peritoneal dialysis.

BACKGROUND: Patients with chronic renal failure have an increased risk of tuberculosis (TB). This occurs with much higher frequency within the first 12 months of initiating dialysis and is usually extrapulmonary in nature. Patients most at risk are those from susceptible ethnic groups, especially the Indian subcontinent. Peritoneal TB, otherwise relatively uncommon, has emerged as an important form of TB in patients undergoing continuous ambulatory peritoneal dialysis (CAPD). METHODS: All cases of peritoneal TB occurring at our institution in patients undergoing CAPD over a 13 year period were identified and analysed. RESULTS: Eight cases were identified, of which seven were non-Caucasian. These patients' characteristics and outcomes are presented. All were undergoing CAPD and most developed TB within 12 months of initiating dialysis. All presented with fever, but symptoms and signs were indistinguishable from bacterial peritonitis. Six were culture-positive, mainly from peritoneal dialysis fluid, but only two cases proved smear-positive. All were treated with standard anti-tuberculous chemotherapy. Three went on to permanent haemodialysis as a result of peritonitis and three have died, one of these as a result of TB. CONCLUSIONS: Peritoneal TB, whilst otherwise relatively uncommon, is an important manifestation of TB in CAPD patients and usually develops soon after commencing dialysis. The reasons for this are unknown and require further research.

The Lac repressor: a second generation of structural and functional studies.

In the past year, the crystal structure of a dimeric version of the Escherichia coli Lac repressor bound to operator DNA was determined at 2.6A resolution, providing a closer view of the operator-bound conformation of the repressor. Refined NMR studies of the DNA-binding portion of the repressor complexed to operator DNA have revealed further details of the unique DNA-binding interactions of the repressor. The structural studies have been complemented by continued biochemical studies, with the overall goal of understanding the mechanism of allosteric regulation.

Crystal structure of the lambda repressor C-terminal domain provides a model for cooperative operator binding.

Interactions between transcription factors bound to separate operator sites commonly play an important role in gene regulation by mediating cooperative binding to the DNA. However, few detailed structural models for understanding the molecular basis of such cooperativity are available. The c1 repressor of bacteriophage lambda is a classic example of a protein that binds to its operator sites cooperatively. The C-terminal domain of the repressor mediates dimerization as well as a dimer-dimer interaction that results in the cooperative binding of two repressor dimers to adjacent operator sites. Here, we present the x-ray crystal structure of the lambda repressor C-terminal domain determined by multiwavelength anomalous diffraction. Remarkably, the interactions that mediate cooperativity are captured in the crystal, where two dimers associate about a 2-fold axis of symmetry. Based on the structure and previous genetic and biochemical data, we present a model for the cooperative binding of two lambda repressor dimers at adjacent operator sites.

A closer view of the conformation of the Lac repressor bound to operator.

Crystal structures of the Lac repressor, with and without isopropyithiogalactoside (IPTG), and the repressor bound to operator have provided a model for how the binding of the inducer reduces the affinity of the repressor for the operator. However, because of the low resolution of the operator-bound structure (4.8 A), the model for the allosteric transition was presented in terms of structural elements rather than in terms of side chain interactions. Here we have constructed a dimeric Lac repressor and determined its structure at 2.6 A resolution in complex with a symmetric operator and the anti-inducer orthonitrophenylfucoside (ONPF). The structure enables the induced (IPTG-bound) and repressed (operator-bound) conformations of the repressor to be compared in atomic detail. An extensive network of interactions between the DNA-binding and core domains of the repressor suggests a possible mechanism for the allosteric transition.

Unusual conformation of nicotinamide adenine dinucleotide (NAD) bound to diphtheria toxin: a comparison with NAD bound to the oxidoreductase enzymes.

The conformation of NAD bound to diphtheria toxin (DT), an ADP-ribosylating enzyme, has been compared to the conformations of NAD(P) bound to 23 distinct NAD(P)-binding oxidoreductase enzymes, whose structures are available in the Brookhaven Protein Data Bank. For the oxidoreductase enzymes, NAD(P) functions as a cofactor in electron transfer, whereas for DT, NAD is a labile substrate in which the N-glycosidic bond between the nicotinamide ring and the N-ribose is cleaved. All NAD(P) conformations were compared by (1) visual inspection of superimposed molecules, (2) RMSD of atomic positions, (3) principal component analysis, and (4) analysis of torsion angles and other conformational parameters. Whereas the majority of oxidoreductase-bound NAD(P) conformations are found to be similar, the conformation of NAD bound to DT is found to be unusual. Distinctive features of the conformation of NAD bound to DT that may be relevant to DT's function as an ADP-ribosylating enzyme include (1) an unusually short distance between the PN and N1N atoms, reflecting a highly folded conformation for the nicotinamide mononucleotide (NMN) portion of NAD, and (2) a torsion angle chi N approximately 0 degree about the scissile N-glycosidic bond, placing the nicotinamide ring outside of the preferred anti and syn orientations. In NAD bound to DT, the highly folded NMN conformation and torsion angle chi N approximately 0 degree could contribute to catalysis, possibly by orienting the C1'N atom of NAD for nucleophilic attack, or by placing strain on the N-glycosidic bond, which is cleaved by DT. The unusual overall conformation of NAD bound to DT is likely to reflect the structure of DT, which is unusual among NAD(P)-binding enzymes. In DT, the NAD binding site is formed at the junction of two antiparallel beta-sheets. In contrast, although the 24 oxidoreductase enzymes belong to at least six different structural classes, almost all of them bind NAD(P) at the C-terminal end of a parallel beta-sheet. The structural alignments and principal component analysis show that enzymes of the same structural class bind to particularly similar conformations of NAD(P), with few exceptions. The conformation of NAD bound to DT superimposes closely with that of an NAD analogue bound to Pseudomonas exotoxin A, an ADP-ribosylating toxin that is structurally homologous to DT. This suggests that all of the ADP-ribosylating enzymes that are structurally homologous to DT and ETA will bind a highly similar conformation of NAD.

Crystal structure of diphtheria toxin bound to nicotinamide adenine dinucleotide.

The crystal structure of diphtheria toxin (DT) in complex with nicotinamide adenine dinucleotide (NAD) has been determined by x-ray crystallography to 2.3 A resolution. NAD binds to a cleft on the surface of the catalytic (C) domain of DT, interacting closely with the side chains of Tyr54, Tyr65, His21, Thr23, and Glu 48. The carboxylate group of Glu148 of Dt lies approximately 4 A from the scissile, N-glycosidic bound of NAD, suggesting a possible catalytic role for Glu148 in stabilizing a positively charged oxocarbonium intermediate. Residues 39-46 of the active-site loop of the C-domain become disordered upon NAD-binding, suggesting a potential role for these residues in binding to elongation facor-2 (EF-2). Structural alignments of the DT-NAD complex with the structures of other ADP-ribosylating toxins suggest how NAD may bind to these other enzymes.

Crystal structure of nucleotide-free diphtheria toxin.

The crystal structure of diphtheria toxin (DT) in the absence of nucleotide (nucleotide-free DT) has been determined at 2.3 A resolution to a crystallographic R factor and free R factor of 18.2 and 28.2%, respectively. A comparison of this structure to the previously determined structures of DT in complex with adenyly(3'-5')uridine monophosphate (ApUp) and DT in complex with nicotinamide adenine dinucleotide (NAD) reveals that there are no significant movements of the two subdomains of the catalytic (C) domain associated with dinucleotide binding. The side chains of six residues within the active-site cleft, including Tyr65, Pro38, Tyr27, Thr23, Glu148, and Tyr54, show movements of up to 3 A upon dinucleotide binding. In the structure of nucleotide-free DT, the active-site loop residues 39-47 of the C domain are well ordered and extend over the active-site cleft in approximately the same position as in the structure of DT in complex with ApUp. This is in contrast to the structure of the DT-NAD complex, in which the active-site loop is disordered. On the basis of a comparison of the nucleotide-free and NAD-bound DT structures, we suggest that the interaction of NAD with Pro38 and also possibly Tyr54 and Trp153 could disrupt the network of hydrogen bonds that stabilizes the position of the active-site loop over the active-site cleft, allowing this loop to become disordered. This may be an important step in binding of the C domain of DT to its substrate, elongation factor-2.

Oligomerization of a 45 kilodalton fragment of diphtheria toxin at pH 5.0 to a molecule of 20-24 subunits.

Diphtheria toxin (DT) is a 58 kDa protein, secreted by lysogenic strains of Corynebacterium diphtheriae, that causes the disease diphtheria in humans. The catalytic (C) domain of DT kills host cells by gaining entry into the cytoplasm and inhibiting protein synthesis. The translocation of the C domain across the endosomal membrane and into the cytoplasm of a host cell is mediated by the translocation (T) domain of DT. This process is triggered by acidification from pH approximately 7 to pH approximately 5 within the endosome. Here we show that crm45 (cross-reacting material of 45 kDa), a 45 kDa deletion mutant of DT which contains the C and T domains but lacks the C-terminal receptor-binding (R) domain, undergoes a transition from a monomer to a large oligomer upon acidification from pH 7.0 to pH 5.0. Dynamic light scattering analysis of crm45 at pH 5.0 results in a polydispersity value of only 8-17%, suggesting that the oligomer is uniformly sized. Using analytical ultracentrifugation, measurements of the sedimentation rate and diffusion coefficient of crm45 at pH 5.0 result in a molecular mass determination of 890 +/- 40 kDa (20 +/- 1 subunits) for the oligomer. Equilibrium sedimentation data on crm45 at pH 5.0 are best fit by a single species with a mass of 1000 +/- 50 kDa (24 +/- 1 subunits). These results reveal the pH-dependent formation of a uniformly sized, 20-24 subunit oligomer of the C and T domains of DT, in solution. Because the oligomer of crm45 forms at the pH of the acidified endosome, it could be relevant to the translocation of the C domain of DT across the endosomal membrane and into the cytoplasm of host cells. The possible relevance of this oligomer of crm45 to the membrane translocation of the C domain of DT correlates with earlier kinetic studies of DT intoxication of Vero cells, which inferred the transfer of approximately 20 C domains of DT to the cytoplasm of host cells, in a single event.

Crystal structure of diphtheria toxin bound to nicotinamide adenine dinucleotide.

Diphtheria toxin (DT), a 58 kDa protein secreted by lysogenic strains of Corynebacterium diphtheriae, causes the disease diphtheria in humans by gaining entry into the cytoplasm of cells and inhibiting protein synthesis. Specifically, the catalytic (C) domain of DT transfers the ADP-ribose group of NAD to elongation factor-2 (EF-2), rendering EF-2 inactive. In order to investigate how the C-domain of DT binds NAD and catalyzes the ADP-ribosylation of EF-2, the crystal structure of DT in complex with NAD has been determined to 2.3 A resolution. This is the first crystal structure of an ADP-ribosyltransferase (ADP-RT) enzyme in complex with NAD and suggests the features of the ADP-RT fold which are important for NAD binding. The conformation of NAD in the complex and the proximity of the Glu148 carboxylate group of the C-domain to the scissile, N-glycosidic bond of NAD suggest plausible modes of catalysis of the ADP-ribosylation reaction. Residues 39-46 of the active-site loop of the C-domain become disordered upon NAD binding, suggesting a potential role for this loop in the recognition of the ADP-ribose acceptor substrate, EF-2. The negatively charged phosphates and two ribose hydroxyls of NAD are not in direct contact with any atoms of the C-domain. Instead, they form an exposed surface which appears to be presented for recognition by EF-2. Structural alignments of the DT-NAD complex with the structures of other members of the ADP-RT family suggest how NAD may bind to these other enzymes.

A comparison of the effects of phenelzine treatment with moclobemide treatment on cardiovascular reflexes.

The effects of treatment with phenelzine (n = 15) and moclobemide (n = 20) on heart rate variability and cardiovascular responses to standing were examined using non-invasive beat-to-beat blood pressure (BP) monitoring in an open cross-sectional study. Phenelzine markedly impaired the BP response compared with moclobemide, with 83% vs 15% of patients lacking the normal initial BP overshoot (p < 0.01). BP recovery to supine levels was delayed (median time for diastolic BP 14.5 s after phenelzine vs 4.9 s after moclobemide; p < 0.002). Standing BP at 1 min and its change from supine levels were also significantly lower in the phenelzine group (delta diastolic BP 4 mmHg vs 15 mmHg; p < 0.001). Heart rate responses and variability were preserved and did not differ between treatments. These findings are consistent with impairment of sympathetic function but preservation of parasympathetic responses after phenelzine treatment.