Rabies outbreak in African Wild Dogs (Lycaon pictus) in the Tuli region, Botswana: Interventions and management mitigation recommendations.

We describe an outbreak of rabies in a pack of African wild dogs (Lycaon pictus) in the Limpopo-Lipadi Private Game and Wilderness Reserve in the Tuli region of south-eastern Botswana. We define the pack's behavioural response to the disease, clinical signs, and management interventions undertaken and make recommendations to mitigate against future disease outbreaks of this nature. The outbreak, which occurred in late 2014 and early 2015, resulted in the death or disappearance of 29 individuals out of a pack of 35 wild dogs. The disruption to the social structure within the pack, the behaviour of the animals and clinical signs were similar to that documented during previous rabies outbreaks amongst African wild dogs in Southern and East Africa in recent years. Management interventions taken during the outbreak were aimed at preventing extirpation of the pack and reducing the risk of further disease spread to other mammals in the reserve.

Exploring the resilience of wheat crops grown in short rotations through minimising the build-up of an important soil-borne fungal pathogen.

Given the increasing demand for wheat which is forecast, cropping of wheat in short rotations will likely remain a common practice. However, in temperate wheat growing regions the soil-borne fungal pathogen Gaeumannomyces tritici becomes a major constraint on productivity. In cultivar rotation field experiments on the Rothamsted Farm (Hertfordshire, UK) we demonstrated a substantial reduction in take-all disease and grain yield increases of up to 2.4 tonnes/ha when a low take-all inoculum building wheat cultivar was grown in the first year of wheat cropping. Phenotyping of 71 modern elite wheat cultivars for the take-all inoculum build-up trait across six diverse trial sites identified a few cultivars which exhibited a consistent lowering of take-all inoculum build-up. However, there was also evidence of a significant interaction effect between trial site and cultivar when a pooled Residual Maximum Likelihood (REML) procedure was conducted. There was no evidence of an unusual rooting phenotype associated with take-all inoculum build-up in two independent field experiments and a sand column experiment. Together our results highlight the complex interactions between wheat genotype, environmental conditions and take-all inoculum build-up. Further work is required to determine the underlying genetic and mechanistic basis of this important phenomenon.

Take-all or nothing.

Take-all disease of Poaceae is caused by Gaeumannomyces graminis (Magnaporthaceae). Four varieties are recognised in G. graminis based on ascospore size, hyphopodial morphology and host preference. The aim of the present study was to clarify boundaries among species and varieties in Gaeumannomyces by combining morphology and multi-locus phylogenetic analyses based on partial gene sequences of ITS, LSU, tef1 and rpb1. Two new genera, Falciphoriella and Gaeumannomycella were subsequently introduced in Magnaporthaceae. The resulting phylogeny revealed several cryptic species previously overlooked within Gaeumannomyces. Isolates of Gaeumannomyces were distributed in four main clades, from which 19 species could be delimited, 12 of which were new to science. Our results show that the former varieties Gaeumannomyces graminis var. avenae and Gaeumannomyces graminis var. tritici represent species phylogenetically distinct from G. graminis, for which the new combinations G. avenae and G. tritici are introduced. Based on molecular data, morphology and host preferences, Gaeumannomyces graminis var. maydis is proposed as a synonym of G. radicicola. Furthermore, an epitype for Gaeumannomyces graminis var. avenae was designated to help stabilise the application of that name.

An analysis of Pseudomonas genomic diversity in take-all infected wheat fields reveals the lasting impact of wheat cultivars on the soil microbiota.

Manipulation of the soil microbiota associated with crop plants has huge promise for the control of crop pathogens. However, to fully realize this potential we need a better understanding of the relationship between the soil environment and the genes and phenotypes that enable microbes to colonize plants and contribute to biocontrol. A recent 2 years of investigation into the effect of wheat variety on second year crop yield in the context of take-all fungal infection presented the opportunity to examine soil microbiomes under closely defined field conditions. Amplicon sequencing of second year soil samples showed that Pseudomonas spp. were particularly affected by the wheat cultivar grown in year one. Consequently, 318 rhizosphere-associated Pseudomonas fluorescens strains were isolated and characterized across a variety of genetic and phenotypic traits. Again, the wheat variety grown in the first year of the study was shown to exert considerable selective pressure on both the extent and nature of Pseudomonas genomic diversity. Furthermore, multiple significant correlations were identified within the phenotypic/genetic structure of the Pseudomonas population, and between individual genotypes and the external wheat field environment. The approach outlined here has considerable future potential for our understanding of plant-microbe interactions, and for the broader analysis of complex microbial communities.

The design and synthesis of potent inhibitors of hepatitis C virus NS3-4A proteinase.

Hepatitis C virus (HCV) is the cause of the majority of transfusion-associated hepatitis and a significant proportion of community-acquired hepatitis worldwide. Infection by HCV frequently leads to persistent infections that result in a range of clinical conditions including an asymptomatic carrier state, severe chronic active hepatitis, cirrhosis and, in some cases, hepatocellular carcinoma. The HCV genome consists of a single-stranded, positive sense RNA containing an open reading frame of approximately 9060 nucleotides. This is translated into a single polyprotein of approximately 3020 amino acids (C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B), which in turn is processed by a series of host and viral proteinases into at least 10 cleavage products. The N-terminal portion of the NS3 protein encodes a serine proteinase that is responsible for the cleavage at the NS3-4A, NS4A-4B, NS4B-5A and NS5A-5B junctions. The 54 amino acid NS4A protein is a cofactor that binds to the NS3 protein and enhances its proteolytic activity. This report describes the expression of a recombinant NS3-4A proteinase fusion protein in Escherichia coli and the in vitro characterization of the enzyme activity using synthetic peptide substrates. It then demonstrates how these results were employed to guide the design of potent inhibitors of this enzyme.

In vitro activity of hepatitis B virus polymerase: requirement for distinct metal ions and the viral epsilon stem-loop.

Hepadnaviruses have a complex replication cycle which includes reverse transcription of the pregenomic RNA. The initial step in this process in hepatitis B virus (HBV) requires the viral polymerase to engage a highly stable region of secondary structure within the pregenomic RNA termed the epsilon stem-loop. While reverse transcriptases belonging to the retrovirus family use a specific cellular tRNA as primer, HBV polymerase utilizes a tyrosine residue located within its own N terminus. Therefore, the first deoxyribonucleotide is covalently coupled to HBV polymerase prior to extension of the DNA strand by conventional reverse transcription. We have expressed HBV polymerase in a baculovirus and following purification have found it to be active with respect to protein-priming and reverse transcription of copurified RNA. Importantly, we found both of these processes to be critically dependent on the presence of the epsilon stem-loop. The metal ion preferences of HBV polymerase were also investigated for both the protein-priming and reverse transcription activities of this enzyme. Reverse transcription was dependent on magnesium, with an optimal concentration of 5 mM. However, protein-priming was strongly favoured by manganese ions and was optimal at a concentration of 1 mM. Thus, using manganese as sole source of metal ions our activity assay is restricted to the protein-priming event and will allow the search for novel antivirals specifically blocking this unique mechanism.

Protein interactions in the herpes simplex virus type 1 VP16-induced complex: VP16 peptide inhibition and mutational analysis of host cell factor requirements.

The herpes simplex virus VP16 protein functions as a potent transcriptional activator and targets DNA sites with the consensus TAATGARAT present in all the viral immediate-early gene promoters. To do so, VP16 directs assembly of a multiprotein complex involving two cellular proteins, host cell factor (HCF) and the Oct-1 DNA-binding transcription factor. To investigate the importance of specific protein-protein interactions to formation of this VP16-induced complex (VIC), we used oligopeptides to prevent VIC assembly. Linear and cyclic peptides corresponding to a region of VP16 previously implicated in complex formation were potent inhibitors of VIC assembly. To further characterize the protein interactions involved, we cloned a human cDNA encoding the minimal VP16 interaction domain of HCF, containing amino acids 1 to 380 [HCF (1-380)]. The REHAYS-based peptides active in preventing VIC assembly were found to specifically block binding of VP16 to HCF (1-380), without affecting VP16-Oct-1 binding. The inhibitory activity of these VP16 peptides was strictly sequence specific for the EHAY residues. Site-directed mutagenesis of the HCF (1-380) domain revealed residues E102 and K105 to be critical determinants in support of VIC formation. Alteration of a single residue in HCF, K105, was shown to virtually abolish complex assembly. Interestingly however, none of the HCF mutants that were impaired in their ability to support complex formation exhibited defects in direct VP16 binding, supporting loss of function at a higher order in complex assembly.

Comparative study of abdominal incision techniques.

The characteristics of surgical incisions made with an electrosurgical technique were compared with those made using conventional methods in a prospective randomized trial. In particular, the claim that the method of incision may influence postoperative pain was investigated. A total of 101 consecutive patients receiving full-length midline laparotomy incisions for gastrointestinal resection were studied. A record was kept of the time required to make the incision and blood loss as well as postoperative pain (using a linear analogue scale), ventilatory function and requirement for analgesia. There were 50 patients in group 1 (scalpel; 15 men, 35 women) and 51 in group 2 (electrocautery; 26 men, 25 women). The groups were similar in age, body-weight, diagnosis and the type of surgical procedures being performed. Incision time was similar in the two groups but median blood loss during incision was significantly less in group 2 patients than in group 1 (10 versus 25 ml, P < 0.0001). Linear analogue pain scores were not significantly different between the groups at any stage after operation. The same was true of postoperative ventilatory function and requirement for analgesia. A total value for morphine use during the entire postoperative period was derived for each group and the median was 1.55 mg/kg for group 1 compared with 1.49 mg/kg for group 2. The electrosurgical method is associated with less blood loss during incision, although this study has failed to confirm any reduction in postoperative pain or requirement for analgesia in these patients.

The effect of habitual sustained endurance exercise on cardiac structure and function.

We performed echocardiographic evaluation of 20 male veteran endurance runners (mean age 56 +/- 7 years) and compared them with a group of age-matched controls. All the runners had run at least 25 miles per week for more than 25 years. Although only four athletes showed voltage ECG evidence of left ventricular hypertrophy, using echocardiography, 10 athletes had evidence of left ventricular hypertrophy. Thus electrocardiography is insufficiently sensitive to detect LVH in this group (10%). Left ventricular posterior wall thickness and left ventricular mass were significantly greater in the athletes, the difference becoming more marked when LV mass was corrected for body surface area and, consequently, the wall thickness: cavity diameter ratio was increased (P less than 0.04). There was no difference between the two groups in left ventricular performance and no correlation between left ventricular mass and running ability. Thus, veteran runners develop structural cardiac changes similar to younger power-trained athletes and do not show characteristics normally expected of 'volume overload' which is thought to occur in running.

Electrocardiographic findings in male veteran endurance athletes.

Twenty male veteran endurance runners and 20 controls underwent resting, exercise, and ambulatory electrocardiography. Four athletes and three controls satisfied voltage criteria for left ventricular hypertrophy. The PR interval was longer in the athletes and they had longer mean (SD) treadmill exercise times (19 (4) v 16 (2) min) than the controls. Four athletes but no controls had greater than 2 mm downsloping ST segment depression during exercise. During 48 hour ambulatory electrocardiography the athletes had a consistently lower heart rate but maintained a circadian variation. Profound bradycardia (less than 35 beats/min) occurred in eight athletes but only one control. Eight athletes and two controls had asystolic pauses ranging from 1.8 to 15 seconds. Six athletes had first degree heart block, four had Mobitz II second degree block, and three had complete heart block. Most conduction abnormalities occurred at night and resolved during exercise. Ventricular ectopic activity was not significantly different between the groups. Thus heart block patterns and profound bradycardia are more frequent in older athletes than their youthful counterparts.

Acute appendicitis in the elderly: a 5-year retrospective study.

Acute appendicitis in elderly patients has not been studied extensively in the United Kingdom. Studies were performed prior to the regular use of broad spectrum antibiotics and tended to include younger patients. We studied retrospectively all elderly patients (n = 30, mean age = 72 years) who presented in the years 1980-85 and a younger group (n = 30, mean age = 23 years) who had a confirmed pathological diagnosis of appendicitis. We found a significant reduction in accurate pre-operative diagnoses, a longer duration of hospital stay and increased complication and mortality rates in the elderly group. Our study suggests that diagnostic accuracy, mortality and morbidity have not improved in comparison to earlier overseas studies.

A case of intravenous and oral mefenamic acid poisoning.

A 22 year old heroin addict was admitted with tonic-clonic seizures, confusion and agitation 10 hours after taking mefenamic acid 5 grams orally and 2.25 grams intravenously. This appears to be the first recorded case of intravenous mefenamic acid abuse and, although not fatal, is a cause of concern. This is a commonly used drug and its seizure inducing potential is well recognised. It may therefore be worthwhile considering the possibility of intravenous abuse of mefenamic acid in heroin addicts admitted with confusion or seizures.

Failure to diagnose the milk-alkali syndrome.

A case of recurrent hypercalcaemia due to chronic continuous ingestion of a calcium-containing antacid is described in whom the diagnosis was delayed because of an insufficiently detailed drug history. Specific named enquiry must be made of all the various calcium-containing drugs when seeking a history of excessive calcium intake if the diagnosis of the milk-alkali syndrome is not to be missed.