Molecular basis for cA6 synthesis by a type III-A CRISPR-Cas enzyme and its conversion to cA4 production.

The type III-A (Csm) CRISPR-Cas systems are multi-subunit and multipronged prokaryotic enzymes in guarding the hosts against viral invaders. Beyond cleaving activator RNA transcripts, Csm confers two additional activities: shredding single-stranded DNA and synthesizing cyclic oligoadenylates (cOAs) by the Cas10 subunit. Known Cas10 enzymes exhibit a fascinating diversity in cOA production. Three major forms-cA3, cA4 and cA6have been identified, each with the potential to trigger unique downstream effects. Whereas the mechanism for cOA-dependent activation is well characterized, the molecular basis for synthesizing different cOA isoforms remains unclear. Here, we present structural characterization of a cA6-producing Csm complex during its activation by an activator RNA. Analysis of the captured intermediates of cA6 synthesis suggests a 3'-to-5' nucleotidyl transferring process. Three primary adenine binding sites can be identified along the chain elongation path, including a unique tyrosine-threonine dyad found only in the cA6-producing Cas10. Consistently, disrupting the tyrosine-threonine dyad specifically impaired cA6 production while promoting cA4 production. These findings suggest that Cas10 utilizes a unique enzymatic mechanism for forming the phosphodiester bond and has evolved distinct strategies to regulate the cOA chain length.

Recruitment of a Middling Promiscuous Enzyme Drives Adaptive Metabolic Evolution in Escherichia coli.

A key step in metabolic pathway evolution is the recruitment of promiscuous enzymes to perform new functions. Despite the recognition that promiscuity is widespread in biology, factors dictating the preferential recruitment of one promiscuous enzyme over other candidates are unknown. Escherichia coli contains four sugar kinases that are candidates for recruitment when the native glucokinase machinery is deleted-allokinase (AlsK), manno(fructo)kinase (Mak), N-acetylmannosamine kinase (NanK), and N-acetylglucosamine kinase (NagK). The catalytic efficiencies of these enzymes are 103- to 105-fold lower than native glucokinases, ranging from 2,400 M-1 s-1 for the most active candidate, NagK, to 15 M-1 s-1 for the least active candidate, AlsK. To investigate the relationship between catalytic activities of promiscuous enzymes and their recruitment, we performed adaptive evolution of a glucokinase-deficient E. coli strain to restore glycolytic metabolism. We observed preferential recruitment of NanK via a trajectory involving early mutations that facilitate glucose uptake and amplify nanK transcription, followed by nonsynonymous substitutions in NanK that enhance the enzyme's promiscuous glucokinase activity. These substitutions reduced the native activity of NanK and reduced organismal fitness during growth on an N-acetylated carbon source, indicating that enzyme recruitment comes at a cost for growth on other substrates. Notably, the two most active candidates, NagK and Mak, were not recruited, suggesting that catalytic activity alone does not dictate evolutionary outcomes. The results highlight our lack of knowledge regarding biological drivers of enzyme recruitment and emphasize the need for a systems-wide approach to identify factors facilitating or constraining this important adaptive process.

Biochemical methods to map and quantify allosteric motions in human glucokinase.

Allosteric regulation of protein function is ubiquitous in biology. Allostery originates from ligand-mediated alterations in polypeptide structure and/or dynamics, which produce a cooperative kinetic or thermodynamic response to changing ligand concentrations. Establishing a mechanistic description of individual allosteric events requires both mapping the relevant changes in protein structure and quantifying the rates of differential conformational dynamics in the absence and presence of effectors. In this chapter, we describe three biochemical approaches to understand the dynamic and structural signatures of protein allostery using the well-established cooperative enzyme glucokinase as a case study. The combined application of pulsed proteolysis, biomolecular nuclear magnetic resonance spectroscopy and hydrogen-deuterium exchange mass spectrometry offers complementary information that can used to establish molecular models for allosteric proteins, especially when differential protein dynamics are involved.

Diabetes mellitus is not a predictor of poor TB treatment outcomes.

OBJECTIVE: To investigate whether diabetes mellitus (DM) influences TB treatment outcomes.METHODS: This was a retrospective observational cohort study of all notified TB cases from a large London TB centre over a 5-year period. WHO criteria were used to define TB treatment outcomes.RESULTS: The prevalence of DM at TB treatment initiation was 15% (126/838). Most patients (83.3%, 105/126) were on hypoglycaemic treatment and well-controlled (median glycated haemoglobin 53.5 mmol/mol). DM patients were older, more likely to be of Asian ethnicity and had a higher pre-treatment weight. Time from presentation to treatment initiation was longer (median 87.5 vs. 63 days; P < 0.001), while they were significantly more comorbid (median Charlson Comorbidity Index 3 vs. 0; P < 0.001). Overall, favourable treatment outcomes were recorded for 89.5% of patients (87.7% vs. 89.8% for DM and non-DM patients respectively, P = 0.52). In multivariable analysis, DM was not associated with unfavourable TB treatment outcomes (OR 0.49, 95% CI 0.23-1.04, P = 0.06). Independent predictors of unfavourable outcome included age, cavitation, chronic neurological disease and malignant neoplasm.CONCLUSIONS: In a well-resourced setting, with predominantly well-controlled DM patients on treatment, DM was not an independent predictor of unfavourable TB treatment outcomes.

COVID-19 vaccination intent among London healthcare workers.

BACKGROUND: The 10-month timeline from conception to regulatory approval of the Pfizer-BioNTech vaccine against SARS-CoV-2 is unprecedented in modern medicine. However, the climate of the pandemic has also seen anti-vaccination sentiments flourish. AIMS: To determine the intent to accept COVID-19 vaccination among healthcare workers at a London Hospital Trust and examine variation in uptake between demographic groups. METHODS: We conducted a cross-sectional survey open to staff working at the trust. Staff rated on a five-point scale the likelihood of them accepting COVID-19 vaccination. RESULTS: We received 514 responses, representing 16% of the workforce. About 59% of staff intended to seek vaccination, 24% to reject and 17% were unsure. There was significantly reduced intended uptake in females, younger age groups, healthcare assistants, nurses, staff of black ethnic backgrounds and those who rejected influenza vaccination. Safety was the dominant concern. CONCLUSIONS: Our study finds COVID-19 vaccinate hesitancy is prevalent among healthcare workers at a London Hospital Trust. It is particularly concerning that hesitancy was highest amongst groups most exposed to COVID-19 and most at risk of severe disease. Reasons behind disparities in uptake must be addressed to protect staff and prevent deepening inequalities within the healthcare workforce.

The Chemosensory Repertoire of the Eastern Diamondback Rattlesnake (Crotalus adamanteus) Reveals Complementary Genetics of Olfactory and Vomeronasal-Type Receptors.

Pitviper sensory perception incorporates diverse stimuli through the integration of trichromatic color vision, bifocal heat-sensing, and dual-system chemoperception. Chemoperception, or olfaction, is mediated by chemoreceptors in the olfactory bulb and the vomeronasal organ, but the true genomic complexity of the gene families and their relative contributions is unknown. A full genomic accounting of pitviper chemoperception directly complements our current understanding of their venoms by generating a more complete polyphenic representation of their predatory arsenal. To characterize the genetic repertoire of pitviper chemoperception, we analyzed a full-genome assembly for Crotalus adamanteus, the eastern diamondback rattlesnake. We identified hundreds of genes encoding both olfactory receptors (ORs; 362 full-length genes) and type-2 vomeronasal receptors (V2Rs; 430 full-length genes). Many chemoreceptor genes are organized into large tandem repeat arrays. Comparative analysis of V2R orthologs across squamates demonstrates how gene array expansion and contraction underlies the evolution of the chemoreceptor repertoire, which likely reflects shifts in life history traits. Chromosomal assignments of chemosensory genes identified sex chromosome specific chemoreceptor genes, providing gene candidates underlying observed sex-specific chemosensory-based behaviors. We detected widespread episodic evolution in the extracellular, ligand-binding domains of both ORs and V2Rs, suggesting the diversification of chemoreceptors is driven by transient periods of positive selection. We provide a robust genetic framework for studying pitviper chemosensory ecology and evolution.

Risk factors for severe disease in patients admitted with COVID-19 to a hospital in London, England: a retrospective cohort study.

COVID-19 has caused a major global pandemic and necessitated unprecedented public health restrictions in almost every country. Understanding risk factors for severe disease in hospitalised patients is critical as the pandemic progresses. This observational cohort study aimed to characterise the independent associations between the clinical outcomes of hospitalised patients and their demographics, comorbidities, blood tests and bedside observations. All patients admitted to Northwick Park Hospital, London, UK between 12 March and 15 April 2020 with COVID-19 were retrospectively identified. The primary outcome was death. Associations were explored using Cox proportional hazards modelling. The study included 981 patients. The mortality rate was 36.0%. Age (adjusted hazard ratio (aHR) 1.53), respiratory disease (aHR 1.37), immunosuppression (aHR 2.23), respiratory rate (aHR 1.28), hypoxia (aHR 1.36), Glasgow Coma Scale <15 (aHR 1.92), urea (aHR 2.67), alkaline phosphatase (aHR 2.53), C-reactive protein (aHR 1.15), lactate (aHR 2.67), platelet count (aHR 0.77) and infiltrates on chest radiograph (aHR 1.89) were all associated with mortality. These important data will aid clinical risk stratification and provide direction for further research.

Nanosecond-Timescale Dynamics and Conformational Heterogeneity in Human GCK Regulation and Disease.

Human glucokinase (GCK) is the prototypic example of an emerging class of proteins with allosteric-like behavior that originates from intrinsic polypeptide dynamics. High-resolution NMR investigations of GCK have elucidated millisecond-timescale dynamics underlying allostery. In contrast, faster motions have remained underexplored, hindering the development of a comprehensive model of cooperativity. Here, we map nanosecond-timescale dynamics and structural heterogeneity in GCK using a combination of unnatural amino acid incorporation, time-resolved fluorescence, and 19F nuclear magnetic resonance spectroscopy. We find that a probe inserted within the enzyme's intrinsically disordered loop samples multiple conformations in the unliganded state. Glucose binding and disease-associated mutations that suppress cooperativity alter the number and/or relative population of these states. Together, the nanosecond kinetics characterized here and the millisecond motions known to be essential for cooperativity provide a dynamical framework with which we address the origins of cooperativity and the mechanism of activated, hyperinsulinemia-associated, noncooperative variants.

Effects of peri-mortem infection on the entomofauna of decomposing buried human remains - a metadata analysis.

The role of infectious disease as a cause of death is undeniable. The affect infectious disease may have on decomposition after death is less well established. Furthermore, virtually no information is available regarding the effects of burial conditions in such circumstances, despite that numerous clandestine burials occur each year. Although many aspects of post-mortem pathology are well understood and provide frequent insight in medicolegal investigation, where buried bodies are concerned, there is great variation in the decomposition processes, depending on extrinsic and intrinsic conditions. Criminal burials and hurriedly dug clandestine graves are seldom deeper than 120 cm allowing access to certain invertebrates, excluding others that only develop in unburied bodies. Numerous studies have reported on such clandestine graves with a purpose to facilitate forensic investigation, but our knowledge of decomposition in deeper graves lags behind, despite several often-cited papers of over a century ago. The poor level of detail in deep-grave knowledge is in part due to resource deficiencies and ethical considerations, but in part due to lack of thorough investigation of the data in papers of often cited prior work. To this end, a metadata analysis assessed a paper written by Dr. Murray Galt Motter in 1898, providing detail of 150 disinterment events with linked medical records from City of Washington cemeteries. This paper, written more than a hundred years ago, was largely descriptive and the detailed data provided in a summary table were never fully analysed. The paper is often quoted despite these obvious oversights. The present study revisits this work, applying a frequency statistical analysis conducted using categorical data and chi-squared analysis. This new analysis reveals patterns and relationships so long 'locked-up' within the body of the table and provides greater understanding of the effect of infectious disease on the abundance of species in the entomofauna associated with deeply buried remains. The data confirm that the presence of adipocere (saponification) is detrimental to development of soil entomofauna ((X2 = 6·64, df = 1, p < 0·01)). Some species, in particular Proisotoma sepulcralis (Collembola), Eleusis pallida (Coleoptera) and Conicera tibialis (Diptera), were positively influenced by association with infectious disease cases (p < 0·01) while only Piophila casei (Diptera) demonstrated a negative association (p < 0·05). Furthermore, the presence of peri-mortem infectious disease, while not necessarily a cause of death, influences post-mortem colonisation of the buried body by insects. The abundance of some species is enhanced, suggesting that bacterial burdens enhance decomposition in a manner favourable to insect feeding and hence abundance, by releasing compounds that the entomofauna feeds on.

Biophysical Spandrels form a Hot-Spot for Kosmotropic Mutations in Bacteriophage Thermal Adaptation.

Temperature plays a dominating role in protein structure and function, and life has evolved myriad strategies to adapt proteins to environmental thermal stress. Cellular systems can utilize kosmotropic osmolytes, the products of complex biochemical pathways, to act as chemical chaperones. These extrinsic molecules, e.g., trehalose, alter local water structure to modulate the strength of the hydrophobic effect and increase protein stability. In contrast, simpler genetic systems must rely on intrinsic mutation to affect protein stability. In naturally occurring microvirid bacteriophages of the subfamily Bullavirinae, capsid stability is randomly distributed across the phylogeny, suggesting it is not phylogenetically linked and could be altered through adaptive mutation. We hypothesized that these phages could utilize an adaptive mechanism that mimics the stabilizing effects of the kosmotrope trehalose through mutation. Kinetic stability of wild-type ID8, a relative of ΦX174, displays a saturable response to trehalose. Thermal adaptation mutations in ID8 improve capsid stability and reduce responsiveness to trehalose suggesting the mutations move stability closer to the kosmotropic saturation point, mimicking the kosmotropic effect of trehalose. These mutations localize to and modulate the hydrophobicity of a cavern formation at the interface of phage coat and spike proteins-an evolutionary spandrel. Across a series of genetically distinct phages, responsiveness to trehalose correlates positively with cavern hydrophobicity suggesting that the level of hydrophobicity of the cavern may provide a biophysical gating mechanism constraining or permitting adaptation in a lineage-specific manner. Our results demonstrate that a single mutation can exploit pre-existing, non-adaptive structural features to mimic the adaptive effects of complex biochemical pathways.

A Single Mutation Unlocks Cascading Exaptations in the Origin of a Potent Pitviper Neurotoxin.

Evolutionary innovations and complex phenotypes seemingly require an improbable amount of genetic change to evolve. Rattlesnakes display two dramatically different venom phenotypes. Type I venoms are hemorrhagic with low systemic toxicity and high expression of tissue-destroying snake venom metalloproteinases. Type II venoms are highly neurotoxic and lack snake venom metalloproteinase expression and associated hemorrhagic activity. This dichotomy hinges on Mojave toxin (MTx), a phospholipase A2 (PLA2) based ß-neurotoxin expressed in Type II venoms. MTx is comprised of a nontoxic acidic subunit that undergoes extensive proteolytic processing and allosterically regulates activity of a neurotoxic basic subunit. Evolution of the acidic subunit presents an evolutionary challenge because the need for high expression of a nontoxic venom component and the proteolytic machinery required for processing suggests genetic changes of seemingly little immediate benefit to fitness. We showed that MTx evolved through a cascading series of exaptations unlocked by a single nucleotide change. The evolution of one new cleavage site in the acidic subunit unmasked buried cleavage sites already present in ancestral PLA2s, enabling proteolytic processing. Snake venom serine proteases, already present in the venom to disrupt prey hemostasis, possess the requisite specificities for MTx acidic subunit proteolysis. The dimerization interface between MTx subunits evolved by exploiting a latent, but masked, hydrophobic interaction between ancestral PLA2s. The evolution of MTx through exaptation of existing functional and structural features suggests complex phenotypes that depend on evolutionary innovations can arise from minimal genetic change enabled by prior evolution.

Knowledge, attitudes and practices towards blood donation in Barbados.

OBJECTIVE: To obtain information to devise strategies for a voluntary blood donor mobilisation campaign in Barbados. BACKGROUND: The World Health Organization (WHO) recommends that 100% blood should be collected from voluntary non-remunerated donors (VNRD), yet the majority of blood donations (75%) in Barbados are family/replacement donations. Increasing VNRD is paramount to achieving a safe, reliable blood supply, and understanding the population is a strategy suggested by the WHO to inform donor recruitment and education. METHODS: Participants in Barbados (n = 429) completed a self-administered questionnaire in 2014. The questionnaire comprised 31 questions, including demographics (age, gender, highest educational attainment) and blood donation-related knowledge, attitudes and practices. Analysis of variance, t-test and linear regression were used to analyse data. RESULTS: A total of 53% (n = 219) of participants had previously donated blood; almost half were family/replacement donors, and over one-third (36·2%) were lapsed donors and had not donated within the past 2 years. Knowledge deficits included blood donation requirements, deferral factors and maximum yearly donations. Most participants (79%) were willing to donate with more information. Participants with higher educational attainment and previous donors had higher total knowledge and attitude scores (P < 0·01). Single, female and younger participants were less likely to donate blood (P < 0·05). CONCLUSIONS: Barbados can likely increase voluntary blood donation rates by addressing knowledge deficits through education campaigns and increasing awareness of the need for donation.

Dual allosteric activation mechanisms in monomeric human glucokinase.

Cooperativity in human glucokinase (GCK), the body's primary glucose sensor and a major determinant of glucose homeostatic diseases, is fundamentally different from textbook models of allostery because GCK is monomeric and contains only one glucose-binding site. Prior work has demonstrated that millisecond timescale order-disorder transitions within the enzyme's small domain govern cooperativity. Here, using limited proteolysis, we map the site of disorder in unliganded GCK to a 30-residue active-site loop that closes upon glucose binding. Positional randomization of the loop, coupled with genetic selection in a glucokinase-deficient bacterium, uncovers a hyperactive GCK variant with substantially reduced cooperativity. Biochemical and structural analysis of this loop variant and GCK variants associated with hyperinsulinemic hypoglycemia reveal two distinct mechanisms of enzyme activation. In α-type activation, glucose affinity is increased, the proteolytic susceptibility of the active site loop is suppressed and the (1)H-(13)C heteronuclear multiple quantum coherence (HMQC) spectrum of (13)C-Ile-labeled enzyme resembles the glucose-bound state. In ß-type activation, glucose affinity is largely unchanged, proteolytic susceptibility of the loop is enhanced, and the (1)H-(13)C HMQC spectrum reveals no perturbation in ensemble structure. Leveraging both activation mechanisms, we engineer a fully noncooperative GCK variant, whose functional properties are indistinguishable from other hexokinase isozymes, and which displays a 100-fold increase in catalytic efficiency over wild-type GCK. This work elucidates specific structural features responsible for generating allostery in a monomeric enzyme and suggests a general strategy for engineering cooperativity into proteins that lack the structural framework typical of traditional allosteric systems.

Functional and structural characterization of a eurytolerant calsequestrin from the intertidal teleost Fundulus heteroclitus.

Calsequestrins (CSQ) are high capacity, medium affinity, calcium-binding proteins present in the sarcoplasmic reticulum (SR) of cardiac and skeletal muscles. CSQ sequesters Ca²âº during muscle relaxation and increases the Ca²âº-storage capacity of the SR. Mammalian CSQ has been well studied as a model of human disease, but little is known about the environmental adaptation of CSQ isoforms from poikilothermic organisms. The mummichog, Fundulus heteroclitus, is an intertidal fish that experiences significant daily and seasonal environmental fluctuations and is an interesting study system for investigations of adaptation at the protein level. We determined the full-length coding sequence of a CSQ isoform from skeletal muscle of F. heteroclitus (FCSQ) and characterized the function and structure of this CSQ. The dissociation constant (K(d)) of FCSQ is relatively insensitive to changes in temperature and pH, thus indicating that FCSQ is a eurytolerant protein. We identified and characterized a highly conserved salt bridge network in FCSQ that stabilizes the formation of front-to-front dimers, a process critical to CSQ function. The functional profile of FCSQ correlates with the natural history of F. heteroclitus suggesting that the eurytolerant function of FCSQ may be adaptive.

Evaluation of serovar-independent ELISA antigens of Actinobacillus pleuropneumoniae in pigs following vaccination or experimental challenge with respiratory pathogens and natural A. pleuropneumoniae serovar 1 challenge.

OBJECTIVE: To compare the sensitivity and cross-reactivity of six serological enzyme-linked immunosorbent assays (ELISAs) based on serovar-independent antigens of Actinobacillus pleuropneumoniae (App). DESIGN: An experimental pig trial using direct or natural challenge with App and direct challenge or vaccination using other common respiratory pathogens. PROCEDURE: A 39-kDa outer membrane protein antigen and five recombinant antigens from the ApxIVA gene of App were evaluated. The latter were derived from the ApxIVA N terminus (ApxIVA-N, ApxIVA-NP, ApxIVA-NPS) or C terminus (ApxIVA-C, ApxIVA-CP). Pigs were sampled after direct challenge with App, Pasteurella multocida or Haemophilus parasuis, after vaccination with these organisms and after natural App infection. Clinical and necropsy findings were evaluated. RESULTS: The 39-kDa ELISA had high sensitivity, but cross-reactivity, following P. multocida challenge. ELISAs using ApxIVA N terminus antigens were clearly more sensitive than C terminus antigens for detection of App-induced disease. Although affinity-purified ApxIVA-NP antigen detected marginally more diseased pigs than the -N and -NPS ELISAs, these assays only detected 41-47% of 17 pigs with lung lesions and microbiological evidence of App based on sampling up to 4-5 weeks after natural (13 pigs) or 5 weeks after direct App serovar 1 challenge (4 pigs). CONCLUSIONS: The 39-kDa ELISA readily detects App exposure and infection, but is adversely affected by P. multocida infection. ApxIVA-N-based ELISAs can be used to evaluate the App status of commercial herds, but a proportion of infected and diseased animals are seronegative at 1 month after likely exposure to aerosol transmission of App from clinical cases.

Resurrecting prehistoric parvalbumins to explore the evolution of thermal compensation in extant Antarctic fish parvalbumins.

Parvalbumins (PVs) from Antarctic notothenioid fishes display a pattern of thermal adaptation that likely reflects evolutionary changes in protein conformational flexibility. We have used ancestral sequence reconstruction and homology modeling to identify two amino acid changes that could potentially account for the present thermal sensitivity pattern of Antarctic fish PVs compared with a PV from a theoretical warm-adapted ancestral fish. To test this hypothesis, ancient PVs were resurrected in the lab using PV from the notothenioid Gobionotothen gibberifrons as a platform for introducing mutations comparable to the reconstructed ancestral PV sequences. The wild-type PV (WT) as well as three mutant expression constructs were engineered: lysine 8 to asparagine (K8N), lysine 26 to asparagine (K26N) and a double mutant (DM). Calcium equilibrium dissociation constants (K(d)) versus temperature curves for all mutants were right-shifted, as predicted, relative to that of WT PV. The K(d) values for the K8N and K26N single mutants were virtually identical at all temperatures and showed an intermediate level of thermal sensitivity. The DM construct displayed a full conversion of thermal sensitivity pattern to that of a PV from a warm/temperate-adapted fish. Additionally, the K(d) versus temperature curve for the WT construct revealed greater thermal sensitivity compared with the mutant constructs. Measurements of the rates of Ca(2+) dissociation (k(off)) showed that all mutants generally had slower k(off) values than WT at all temperatures. Calculated rates of Ca(2+) binding (k(on)) for the K8N and K26N mutants were similar to values for the WT PV at all temperatures. In contrast, the calculated k(on) values for the DM PV were faster, providing mechanistic insights into the nature of potentially adaptive changes in Ca(2+) binding in this PV. The overall results suggest that the current thermal phenotype of Antarctic PVs can be recapitulated by just two amino acid substitutions.

Evaluation of serovar-independent ELISA antigens of Actinobacillus pleuropneumoniae in pigs, following experimental challenge with A. pleuropneumoniae, Mycoplasma hyopneumoniae and Pasteurella multocida.

OBJECTIVE: To compare the sensitivity and specificity of six serological enzyme-linked immunosorbent assays (ELISAs) based on serovar-independent antigens of Actinobacillus pleuropneumoniae (App) and investigate cross-reactivity in disease-free pigs challenged with Mycoplasma hyopneumoniae and Pasteurella multocida. DESIGN: Five experimental pig trials using direct challenge with App serovars 1, 7 or 15 or direct challenge with M. hyopneumoniae and/or various dose rates of P. multocida. PROCEDURE: A 39-kDa outer membrane protein antigen and five recombinant antigens from the apxIVA gene of App were evaluated. The latter were derived from the ApxIVA N-terminus (ApxIVA-N, ApxIVA-NP, ApxIVA-NPS) or C-terminus (ApxIVA-C, ApxIVA-CP). Pigs were sampled after challenge and clinical and necropsy findings evaluated. RESULTS: The 39-kDa ELISA had high sensitivity but lacked specificity, with significantly increased cross-reactivity following P. multocida challenge. ELISAs based on ApxIVA N-terminus antigens were significantly more sensitive than C-terminus antigens for the detection of App-induced disease. Although ApxIVA-N and ApxIVA-NP ELISAs had increased reactivity following P. multocida challenge, they retained high specificity for App-induced disease (90-93%). Affinity purified ApxIVA-NP antigen had marginally better specificity than ApxIVA-N, without reduced sensitivity. Mycoplasma hyopneumoniae did not affect serological cross-reactivity. In disease-free pigs, the specificity of the ApxIVA-NPS ELISA may be adversely affected by nasal carriage of apparently low-virulence App strains. CONCLUSIONS: ApxIVA-N-based ELISAs can be used for evaluating App status in commercial herds, but some appear limited by high carriage rates of low-virulence App. The 39-kDa antigen is only of merit in exclusion of App disease by negative serology.

Bacterial contamination of stethoscopes on the intensive care unit.

We assessed how often bedside stethoscopes in our intensive care unit were cleaned and whether they became colonised with potentially pathogenic bacteria. On two separate days the 12 nurses attending the bedspaces were questioned about frequency of stethoscope cleaning on the unit and the bedside stethoscopes were swabbed before and after cleaning to identify colonising organisms. Twenty-two health care providers entering the unit were asked the same questions and had their personal stethoscopes swabbed. All 32 non-medical staff cleaned their stethoscopes at least every day; however only three out of the 12 medical staff cleaned this often. Out of 24 intensive care unit bedside stethoscopes tested, two diaphragms and five earpieces were colonised with pathogenic bacteria. MRSA cultured from one earpiece persisted after cleaning. Three out of the 22 personal stethoscope diaphragms and five earpieces were colonised with pathogens. After cleaning, two diaphragms and two earpieces were still colonised, demonstrating the importance of regular cleaning.

Serological responses to two serovar-independent ELISA antigens of Actinobacillus pleuropneumoniae in Australian commercial pig herds.

OBJECTIVE: To compare serological responses in pig herds classified as low or high risk for disease caused by Actinobacillus pleuropneumoniae, using two ELISA tests based on serovar-independent antigens. PROCEDURE: Cross-sectional sampling was undertaken in 13 commercial herds, the clinical and slaughter histories of which indicated either freedom from (n = 5) or prior confirmed cases of A. pleuropneumoniae (n = 8). In nine herds, approximately 40 pigs each were sampled at 4, 8, 12, 16 and 20 weeks. Three of the remaining four herds were sampled between 6 and 30 weeks of age, and the last was sampled only prior to slaughter, at approximately 24 weeks. Sera were tested in ELISA based on two antigens common among A. pleuropneumoniae serovars: a 39-kDa outer membrane protein and a recombinant ApxIVA-N terminus protein. RESULTS: Sampling of 1 and 5 to 6-month-old pigs provided the most useful information on herd status. The 39-kDa ELISA was sensitive in detecting infected herds, but had evidence of cross-reactivity with high seroreactivity rates in older pigs in some low-risk herds. The ApxIVA-N ELISA was less seroreactive in high-risk herds and had higher specificity in low-risk herds. CONCLUSION: ELISA based on the 39-kDa subunit are of limited use, because of possible cross-reactivity, but a high negative predictive value may be useful for risk assessment in suspect herds. Maternal antibody to ApxIVA-N may be of value in detecting high-risk herds, but 5% of 4-week-old pigs in low-risk herds were also seropositive in this assay.