A Novel, High-sensitivity, Bacteriophage-based Assay Identifies Low-level Mycobacterium tuberculosis Bacteremia in Immunocompetent Patients With Active and Incipient Tuberculosis.

The haematogenous dissemination of Mycobacterium tuberculosis (Mtb) is critical to the pathogenesis of progressive tuberculous infections in animal models. Using a novel, phage-based blood assay, we report the first concordant evidence in well-characterized, immunocompetent human cohorts, demonstrating associations of Mtb bacteremia with progressive phenotypes of latent infection and active pulmonary tuberculosis.

A mixed methods evaluation of got care!

This study aimed to assess the relationship between an Interprofessional Collaborative Practice (IPCP) intervention for community-dwelling older adults, Geriatric Outreach and Training with Care! (GOT Care!), and the observed 26% reduction in Emergency Department (ED) visits for the 51 older adult participants. A convergent parallel mixed-methods design was utilized. Demographic data and ED visit data were collected and analyzed using paired-samples t-tests, poisson regression and generalized poisson regression. Stakeholder perspectives were assessed via emailed open-ended surveys and analyzed using content analysis. The quantitative results were transformed into trends that were compared and contrasted with the qualitative themes. The results were consistent with the current literature that IPCP models may have a greater impact on older adults with certain demographic characteristics such as polypharmacy, diabetes and prior ED use, while nursing was identified as an ideal leader for IPCP teams.

CONSERVATION CHALLENGES: THE LIMITATIONS OF ANTEMORTEM TUBERCULOSIS TESTING IN CAPTIVE ASIATIC LIONS (PANTHERA LEO PERSICA).

Genetic diversity of captive wild animals can be enhanced by moving those individuals with valuable genes between collections and through introduction of a new pair from a range country. This requires movement of animals, which is inherent with disease risks, such as the introduction of pathogenic Mycobacterium sp. (MTBC) into a zoological collection. Decisions need to be made based on the outcome of perimovement disease screening using an array of tests, the majority of which are unvalidated in the species. A pair of endangered Asiatic lions (Panthera leo persica) imported from India to the United Kingdom were screened for MTBC using the comparative intradermal tuberculosis (TB) test, the feline interferon-γ blood test, and the experimental bacteriophage assay. Reactions on all three tests prompted screening of the three resident Asiatic lions using the same tests, all of which were negative for MTBC. Based on these test results, the decision had to be made to exclude the genetically valuable pair from the current collection. MTBC could not be identified using further tests, including culture and PCR on a bronchoalveolar lavage, on feces, or on postmortem tissues. This case series highlights the usefulness of a control group when interpreting unvalidated test results for detection of MTBC, the value of training big cats for conscious blood sampling, and the practical implications of placing the comparative intradermal TB test in the eyelids, when dealing with a species that requires a general anesthetic for most hands-on interventions.

Survival of Mycobacterium avium subspecies paratuberculosis in retail pasteurised milk.

A survey of retail purchased semi-skimmed pasteurised milk (n = 368) for Mycobacterium avium subspecies paratuberculosis (MAP) was conducted between May 2014 and June 2015 across the midlands of England using the Phage-PCR assay. Overall, 10.3% of the total samples collected contained viable MAP cells, confirming that pasteurisation is not capable of fully eliminating human exposure to viable MAP through milk. Comparison of the results gained using the Phage-PCR assay with the results of surveys using either culture or direct PCR suggest that the phage-PCR assay is able to detect lower numbers of cells, resulting in an increase in the number of MAP-positive samples detected. Comparison of viable count and levels of MAP detected in bulk milk samples suggest that MAP is not primarily introduced into the milk by faecal contamination but rather are shed directly into the milk within the udder. In addition results detected an asymmetric distribution of MAP exists in the milk matrix prior to somatic cell lysis, indicating that the bacterial cells in naturally contaminated milk are clustered together and may primarily be located within somatic cells. These latter two results lead to the hypothesis that intracellular MAP within the somatic cells may be protected against heat inactivation during pasteurisation, accounting for the presence of low levels of MAP detected in retail milk.

Evaluation of the limitations and methods to improve rapid phage-based detection of viable Mycobacterium avium subsp. paratuberculosis in the blood of experimentally infected cattle.

BACKGROUND: Disseminated infection and bacteraemia is an underreported and under-researched aspect of Johne's disease. This is mainly due to the time it takes for Mycobacterium avium subsp. paratuberculosis (MAP) to grow and lack of sensitivity of culture. Viable MAP cells can be detected in the blood of cattle suffering from Johne's disease within 48 h using peptide-mediated magnetic separation (PMMS) followed by bacteriophage amplification. The aim of this study was to demonstrate the first detection of MAP in the blood of experimentally exposed cattle using the PMMS-bacteriophage assay and to compare these results with the immune response of the animal based on serum ELISA and shedding of MAP by faecal culture. RESULTS: Using the PMMS-phage assay, seven out of the 19 (37 %) MAP-exposed animals that were tested were positive for viable MAP cells although very low numbers of MAP were detected. Two of these animals were positive by faecal culture and one was positive by serum ELISA. There was no correlation between PMMS-phage assay results and the faecal and serum ELISA results. None of the control animals (10) were positive for MAP using any of the four detection methods. Investigations carried out into the efficiency of the assay; found that the PMMS step was the limiting factor reducing the sensitivity of the phage assay. A modified method using the phage assay directly on isolated peripheral blood mononuclear cells (without PMMS) was found to be superior to the PMMS isolation step. CONCLUSIONS: This proof of concept study has shown that viable MAP cells are present in the blood of MAP-exposed cattle prior to the onset of clinical signs. Although only one time point was tested, the ability to detect viable MAP in the blood of subclinically infected animals by the rapid phage-based method has the potential to increase the understanding of the pathogenesis of Johne's disease progression by warranting further research on the presence of MAP in blood.

State-of-the-art detection of Mycobacterium tuberculosis in blood during tuberculosis infection using phage technology.

Tuberculosis (TB), an aerosol-transmitted infection caused by Mycobacterium tuberculosis (Mtb), remains the commonest cause of death globally, from an infectious bacterial disease. Nine years on from the launch of the World Health Organization (WHO)'s END-TB strategy, disease incidence rates are stubbornly unchanged [1]. While this represents, in part, a reversal of improving trends caused by the COVID-19 pandemic, it also reflects the fragility and inadequacy of healthcare systems to sustain TB control [2]. Although multifactorial, a key reason for this is the ineffectiveness of existing clinical tools to meet the two key objectives of the END-TB strategy-(i) early diagnosis and treatment of TB disease (to limit onward transmission); and (ii) disease prevention through screening for asymptomatic TB infection (TBI). Meeting both objectives will rely on the development of new biomarkers with high accuracy, but the global nature of the TB problem also requires that new tests are rapid, low cost and can be measured in patients by sampling from universally accessible sites. In this review, we will present the accumulating evidence for circulating Mtb in both TB disease and asymptomatic TBI and discuss the potential utility of novel bacteriophage-based technology for blood-based detection of Mtb.

PET-CT-guided characterisation of progressive, preclinical tuberculosis infection and its association with low-level circulating Mycobacterium tuberculosis DNA in household contacts in Leicester, UK: a prospective cohort study.

BACKGROUND: Incipient tuberculosis, a progressive state of Mycobacterium tuberculosis infection with an increased risk of developing into tuberculosis disease, remains poorly characterised. Animal models suggest an association of progressive infection with bacteraemia. Circulating M tuberculosis DNA has previously been detected in pulmonary tuberculosis by use of Actiphage, a bacteriophage-based real-time PCR assay. We aimed to investigate whether serial [18F]fluorodeoxyglucose ([18F]FDG)-PET-CT could be used to characterise the state and progressive trajectory of incipient tuberculosis, and examine whether these PET-CT findings are associated with Actiphage-based detection of circulating M tuberculosis DNA. METHODS: We did a prospective 12-month cohort study in healthy, asymptomatic adults (aged ≥16 years) who were household contacts of patients with pulmonary tuberculosis, and who had a clinical phenotype of latent tuberculosis infection, in Leicester, UK. Actiphage testing of participants' blood samples was done at baseline, and [18F]FDG PET-CT at baseline and after 3 months. Baseline PET-CT features were classified as positive, indeterminate, or negative, on the basis of the quantitation (maximum standardised uptake value [SUVmax]) and distribution of [18F]FDG uptake. Microbiological sampling was done at amenable sites of [18F]FDG uptake. Changes in [18F]FDG uptake after 3 months were quantitatively categorised as progressive, stable, or resolving. Participants received treatment if features of incipient tuberculosis, defined as microbiological detection of M tuberculosis or progressive PET-CT change, were identified. FINDINGS: 20 contacts were recruited between Aug 5 and Nov 5, 2020; 16 of these participants had a positive result on IFNγ release assay (QuantiFERON-TB Gold Plus [QFT]) indicating tuberculosis infection. Baseline PET-CT scans were positive in ten contacts (all QFT positive), indeterminate in six contacts (three QFT positive), and negative in four contacts (three QFT positive). Four of eight PET-CT-positive contacts sampled had M tuberculosis identified (three through culture, one through Xpert MTB/RIF Ultra test) from intrathoracic lymph nodes or bronchial wash and received full antituberculosis treatment. Two further unsampled PET-CT-positive contacts were also treated: one with [18F]FDG uptake in the lung (SUVmax 9·4) received empirical antituberculosis treatment and one who showed progressive [18F]FDG uptake received preventive treatment. The ten untreated contacts with [18F]FDG uptake at baseline (seven QFT positive) had stable or resolving changes at follow-up and remained free of tuberculosis disease after 12 months. A positive baseline Actiphage test was associated with the presence of features of incipient tuberculosis requiring treatment (p=0·018). INTERPRETATION: Microbiological and inflammatory features of incipient tuberculosis can be visualised on PET-CT and are associated with M tuberculosis detection in the blood, supporting the development of pathogen-directed blood biomarkers of tuberculosis risk. FUNDING: MRC Confidence in Concept.

Dead in the water - Role of relic DNA and primer choice for targeted sequencing surveys of anaerobic sewage sludge intended for biological monitoring.

DNA-based monitoring of microbial communities that are responsible for the performance of anaerobic digestion of sewage wastes has the potential to improve resource recoveries for wastewater treatment facilities. By treating sludge with propidium monoazide (PMA) prior to amplicon sequencing, this study explored how the presence of DNA from dead microbial biomass carried over with feed sludge may mislead process-relevant biomarkers, and whether primer choice impacts such assessments. Four common primers were selected for amplicon preparation, also to determine if universal primers have sufficient taxonomic or functional coverage for monitoring ecological performance; or whether two domain-specific primers for Bacteria and Archaea are necessary. Anaerobic sludges of three municipal continuously stirred-tank reactors in Victoria, Australia, were sampled at one time-point. A total of 240 amplicon libraries were sequenced on a Miseq using two universal and two domain-specific primer pairs. Untargeted metabolomics was chosen to complement biological interpretation of amplicon gene-based functional predictions. Diversity, taxonomy, phylogeny and functional potentials were systematically assessed using PICRUSt2, which can predict community wide pathway abundance. The two chosen universal primers provided similar diversity profiles of abundant Bacteria and Archaea, compared to the domain-specific primers. About 16 % of all detected prokaryotic genera covering 30 % of total abundances and 6 % of PICRUSt2-estimated pathway abundances were affected by PMA. This showed that dead biomass in the anaerobic digesters impacted DNA-based assessments, with implications for predicting active processes, such as methanogenesis, denitrification or the identification of organisms associated with biological foams. Hence, instead of running two sequencing runs with two different domain-specific primers, we propose conducting PMA-seq with universal primer pairs for routine performance monitoring. However, dead sludge biomass may have some predictive value. In principal component analysis the compositional variation of 239 sludge metabolites resembled that of 'dead-plus-alive' biomass, suggesting that dead organisms contributed to the potentially process-relevant sludge metabolome.

Impact of volatile solids destruction on the shear and solid-liquid separation behaviour of anaerobic digested sludge.

Systematic and comprehensive characterisation of shear and solid-liquid separation properties of sludge across a wide range of solids concentration and volatile solids destruction (VSD) is critical for design and optimization of the anaerobic digestion process. In addition, there is a need for studies at the psychrophilic temperature range as many unheated anaerobic digestion processes are operated under ambient conditions with minimal self-heating. In this study, two digesters were operated at different combinations of operating temperature (15-25 °C) and hydraulic retention time (16-32 d) to ensure a wide range of VSD in the range of 0.42-0.7 was obtained. For shear rheology, the viscosity increased 1.3 to 3.3 times with the increase of VSD from 43 % to 70 %, while other parameters (temperature, VS fraction) having a negligible impact. Analysis of a hypothetical digester indicated that there is an optimum VSD range 65-80 % where increase in viscosity due to the higher VSD is balanced by the decrease in solids concentration. For solid-liquid separation, a thickener model and a filtration model were used. No significant impact of VSD on the solids flux, underflow solids concentrations or specific solids throughput was observed in the thickener and filtration model. However, there was an increase in average cake solids concentration from 21 % to 31 % with increase of VSD from 55 % to 76 %, indicating better dewatering behaviour.

Shear and solid-liquid separation behaviour of anaerobic digested sludge across a broad range of solids concentrations.

Due to the non-homogeneous and multiphase nature of anaerobic lagoon constituents, CFD modelling for process optimisation requires continuous functions for shear and solid-liquid separation properties across a large range of solids concentrations. Unfortunately, measurement of existing material properties of anaerobic sludges is limited to only shear or solid-liquid separation, or to a limited solids concentration. In this work, the shear properties of an anaerobic sludge were measured from 0.4 to 12.5 vol%, which corresponds to the solids concentrations seen in lagoons. The sludge showed Newtonian behaviour at 0.4 vol% and Herschel-Bulkley yield stress fluid behaviour for higher concentrations ranging from 0.5 to 12 vol%. We compared multiple approaches to determine relationships between the model fitting parameters of consistency, k, flow index, n, and shear yield stress, τy with solids volume fraction ϕ.The solid-liquid separation properties were measured from sedimentation and filtration experiments to obtain compressibility and permeability properties across all the above-mentioned concentrations, enabling development of hindered velocity sedimentation curves. Comparison to full-scale anaerobic digestate identified that the pilot lagoon sludge had faster sedimentation at a given solids concentration in comparison to the digestate. This is the first study on simultaneous rheological characterisation and solid-liquid separation behaviour of an anaerobic sludge across a wide range of concentrations, thus enabling CFD modelling of the hydrodynamics and performance of anaerobic lagoons.

Characterising sedimentation velocity of primary waste water solids and effluents.

Sedimentation in waste water is a heavily studied topic, but mainly focused on hindered and compression settling in secondary sludge, a largely monodispersed solids, where bulk sedimentation velocity is effectively described by functions such as double Vesilind (Takacs). However, many waste water solids, including primary sludge and anaerobic digester effluent are polydispersed, for which application of velocity functions is not well understood. These systems are also subject to large concentration gradients, and poor availability of settling velocity functions has limited design and computational fluid dynamic (CFD) analysis of these units. In this work, we assess the use of various sedimentation functions in single and multi-dimensional domains, comparing model results against multiple batch settling tests at a range of high and low concentrations. Both solids concentration and sludge bed height (interface) over time are measured and compared. The method incorporates uncertainty analysis using Monte Carlo regression, DIRECT (dividing rectangles), and Newton optimisation. It was identified that a double Vesilind (Takacs) model was most effective in the dilute regime (<1%v/v), but could not effectively fit high solids concentrations (>1%v/v) without a substantial (50%) decrease in effective maximum sedimentation velocity (V0). Other parameters (Rh, Rp) did not change. A power law velocity model (Diehl) was significantly less predictive at low concentrations, and not significantly better at higher concentrations. The optimised model (with reduction in V0) was tested vs a standard (optimised) double Vesilind velocity model in a simple primary sedimentation unit, and resulted in deviation from -12% to +18% in solids capture prediction from underload to overload (washout) conditions, indicating that the effect is important in CFD based analysis of these systems.

The role of microbial ecology in improving the performance of anaerobic digestion of sewage sludge.

The use of next-generation diagnostic tools to optimise the anaerobic digestion of municipal sewage sludge has the potential to increase renewable natural gas recovery, improve the reuse of biosolid fertilisers and help operators expand circular economies globally. This review aims to provide perspectives on the role of microbial ecology in improving digester performance in wastewater treatment plants, highlighting that a systems biology approach is fundamental for monitoring mesophilic anaerobic sewage sludge in continuously stirred reactor tanks. We further highlight the potential applications arising from investigations into sludge ecology. The principal limitation for improvements in methane recoveries or in process stability of anaerobic digestion, especially after pre-treatment or during co-digestion, are ecological knowledge gaps related to the front-end metabolism (hydrolysis and fermentation). Operational problems such as stable biological foaming are a key problem, for which ecological markers are a suitable approach. However, no biomarkers exist yet to assist in monitoring and management of clade-specific foaming potentials along with other risks, such as pollutants and pathogens. Fundamental ecological principles apply to anaerobic digestion, which presents opportunities to predict and manipulate reactor functions. The path ahead for mapping ecological markers on process endpoints and risk factors of anaerobic digestion will involve numerical ecology, an expanding field that employs metrics derived from alpha, beta, phylogenetic, taxonomic, and functional diversity, as well as from phenotypes or life strategies derived from genetic potentials. In contrast to addressing operational issues (as noted above), which are effectively addressed by whole population or individual biomarkers, broad improvement and optimisation of function will require enhancement of hydrolysis and acidogenic processes. This will require a discovery-based approach, which will involve integrative research involving the proteome and metabolome. This will utilise, but overcome current limitations of DNA-centric approaches, and likely have broad application outside the specific field of anaerobic digestion.

Effects of liquid type, delivery method, and bolus volume on penetration-aspiration scores in healthy older adults during flexible endoscopic evaluation of swallowing.

OBJECTIVES: The type of liquid (eg, water or milk) that should be used during flexible endoscopic evaluation of swallowing (FEES) has received little investigation. Aspiration may vary as a function of the thin liquid type used during FEES. METHODS: We measured the effects of liquid type (water, skim milk, 2% milk, and whole milk; all dyed with green food coloring), delivery method (cup and straw), and bolus volume (5, 10, 15, and 20 mL) on Penetration-Aspiration Scale (PAS) scores in 14 healthy older adults (mean, 75 years; range, 69 to 85 years). Each participant generated 32 swallows. RESULTS: The PAS scores differed significantly by liquid type (p = 0.003) and by bolus volume (p = 0.017), but not by delivery method (p = 0.442). The PAS scores were significantly greater for 2% milk and whole milk than for skim milk and water (p < 0.05), and for 20 mL versus smaller volumes. Penetration and aspiration were observed on 113 (25%) and 15 (3%) of 448 swallows, respectively. CONCLUSIONS: These findings suggest that both milk and water should be used during FEES for an accurate assessment of aspiration status.

Fluoroscopic surrogate for pharyngeal strength: the pharyngeal constriction ratio (PCR).

The pharyngeal constriction ratio (PCR), derived directly from videofluoroscopy without the need for manometry, requires validation as a surrogate for pharyngeal strength. A correlation of -0.70 was previously identified between PCR and pharyngeal clearing pressures (PP) on separate fluoroscopic and manometric studies. As PP increases, PCR decreases. The objective of the current study was to evaluate the correlation between PCR and PP in 25 patients undergoing simultaneous fluoroscopy and pharyngeal manometry. The effect of the manometric catheter on PCR was also investigated. The correlation between the PCR and averaged pharyngeal clearing pressures was -0.72 (p<0.001). All patients with a PCR>0.25 had a P <60 mmHg. PCR did not differ significantly as a consequence of the manometric catheter. Results suggest the utility of an objective fluoroscopic measure in assessing pharyngeal strength when manometry may not be available or possible.

The effects of aspiration status, liquid type, and bolus volume on pharyngeal peak pressure in healthy older adults.

The reasons for aspiration in healthy adults remain unknown. Given that the pharyngeal phase of swallowing is a key component of the safe swallow, it was hypothesized that healthy older adults who aspirate are likely to generate less pharyngeal peak pressures when swallowing. Accordingly, pharyngeal and upper esophageal sphincter pressures were examined as a function of aspiration status (i.e., nonaspirator vs. aspirator), sensor location (upper vs. lower pharynx), liquid type (i.e., water vs. milk), and volume (i.e., 5 vs. 10 ml) in healthy older adults. Manometric measurements were acquired with a 2.1-mm catheter during flexible endoscopic evaluation. Participants (N = 19, mean age = 79.2 years) contributed 28 swallows; during 8 swallows, simultaneous manometric measurements of upper and lower pharyngeal and upper esophageal pressures were obtained. Pharyngeal manometric peak pressure was significantly less for aspirators (mean = 82, SD = 31 mmHg) than for nonaspirators (mean = 112, SD = 20 mmHg), and upper pharyngeal pressures (mean = 85, SD = 32 mmHg) generated less pressure than lower pharyngeal pressures (mean = 116, SD = 38 mmHg). Manometric measurements vary with respect to aspiration status and sensor location. Lower pharyngeal pressures in healthy older adults may predispose them to aspiration.

Properties of the Extracellular Polymeric Substance Layer from Minimally Grown Planktonic Cells of Listeria monocytogenes.

The bacterium Listeria monocytogenes is a serious concern to food processing facilities because of its persistence. When liquid cultures of L. monocytogenes were prepared in defined media, it was noted that planktonic cells rapidly dropped out of suspension. Zeta potential and hydrophobicity assays found that the cells were more negatively charged (-22, -18, -10 mV in defined media D10, MCDB 202 and brain heart infusion [BHI] media, respectively) and were also more hydrophobic. A SEM analysis detected a capsular-like structure on the surface of cells grown in D10 media. A crude extract of the extracellular polymeric substance (EPS) was found to contain cell-associated proteins. The proteins were removed with pronase treatment. The remaining non-proteinaceous component was not stained by Coomassie blue dye and a further chemical analysis of the EPS did not detect significant amounts of sugars, DNA, polyglutamic acid or any other specific amino acid. When the purified EPS was subjected to attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, the spectra obtained did not match the profile of any of the 12 reference compounds used. An x-ray diffraction (XRD) analysis showed that the EPS was amorphous and a nuclear magnetic resonance (NMR) analysis detected the presence of glycerol. An elemental energy dispersive x-ray (EDX) analysis showed traces of phosphorous as a major component. In conclusion, it is proposed that the non-proteinaceous component may be phospholipid in nature, possibly derived from the cell wall lipoteichoic acid.

Development of a Method to Detect Mycobacterium paratuberculosis in the Blood of Farmed Deer Using Actiphage® Rapid.

Mycobacterium avium subsp paratuberculosis (MAP) is the causative agent of Johne's disease, which is an economically and clinically relevant pathogen for commercial deer production. The purpose of this study was to develop a method that could be used to rapidly detect MAP infection in deer using the Actiphage Rapid blood test. This test has previously been used to detect MAP in cattle blood following the purification of buffy coat using Ficoll gradients, however this method is quite laborious and costly. The purpose of this study was to develop a simpler method of blood preparation that was also compatible with deer blood and the Actiphage test. Initially differential lysis of RBCs using Ammonium Chloride-Potassium (ACK) blood lysis buffer was compared with the Ficoll gradient centrifugation method using cattle blood samples for compatibility with the Actiphage reagents, and it was found that the simpler ACK method did not have an impact on the Actiphage test reagents, producing an equivalent sensitivity for detection of low levels of MAP. When the two methods were compared using clinical blood samples from farmed deer, the ACK lysis method resulted in a cleaner sample. When a blinded test of 132 animals from 4 different production groups was carried out, the majority of the positive test results were found to be from animals in just one group, with a small number identified in a second group. The test results were found to be reproducible when a small set of positive animals were tested again 1 month after their initial testing. Finally a set of negative animals which had been previously screened using an ELISA test, all animals gave a negative Actiphage result. This study shows that this improved sample preparation method and Actiphage blood testing can be used to test blood samples from deer, and the full diagnostic potential of the method can now be evaluated.

Tuberculosis in badgers where the bovine tuberculosis epidemic is expanding in cattle in England.

Bovine tuberculosis (bTB) is an important animal health and economic problem for the cattle industry and a potential zoonotic threat. Wild badgers (Meles meles) play a role on its epidemiology in some areas of high prevalence in cattle, particularly in the UK and Republic of Ireland and increasingly in parts of mainland Europe. However, little is known about the involvement of badgers in areas on the spatial edge of the cattle epidemic, where increasing prevalence in cattle is seen. Here we report the findings of a study of found-dead (mainly road-killed) badgers in six counties on the edge of the English epidemic of bTB in cattle. The overall prevalence of Mycobacterium tuberculosis complex (MTC) infection detected in the study area was 51/610 (8.3%, 95% CI 6.4-11%) with the county-level prevalence ranging from 15 to 4-5%. The MTC spoligotypes of recovered from badgers and cattle varied: in the northern part of the study area spoligotype SB0129 predominated in both cattle and badgers, but elsewhere there was a much wider range of spoligotypes found in badgers than in cattle, in which infection was mostly with the regional cattle spoligotype. The low prevalence of MTC in badgers in much of the study area, and, relative to in cattle, the lower density of sampling, make firm conclusions difficult to draw. However, with the exception of Cheshire (north-west of the study area), little evidence was found to link the expansion of the bTB epidemic in cattle in England to widespread badger infection.

Effects of Gold laser on the avian chorioallantoic membrane.

OBJECTIVES: Office-based lasers have revolutionized the treatment of laryngeal disease. The 980-nm Gold laser is a device that may offer some practical advantages over other office lasers. The chick chorioallantoic membrane has been proposed as a model for predicting the effects of photoangiolytic lasers on vocal fold microvasculature. We sought to evaluate the effects of the Gold laser in this model. METHODS: Vascular reactions in first-order vessels were determined for the Gold laser with both 0 degree straight and 30 degrees angled laser fibers. Vessels were treated at 15 W and a 500-ms pulse interval, with a 1-mm working distance. Pulse widths of 300 ms and 500 ms were evaluated. All vessels were treated until selective coagulation or vessel rupture. RESULTS: We performed 60 trials on 30 embryos. The mean energy delivered was 33.7 J for the straight fiber and 51.2 J for the angled fiber. The laser achieved selective vessel coagulation without rupture in 100% (30 of 30) of straight fiber trials and in 100% (30 of 30) of angled fiber trials. In 6.7% (2 of 30) of straight fiber and 10% (3 of 30) of angled fiber trials, it caused minor injury to the surrounding albumin as indicated by white coagulum outside the vessel. CONCLUSIONS: The Gold laser effectively coagulates small vessels without rupture at a working distance of 1 mm and settings of 15 W, 500-ms pulse interval, and 300- to 500-ms pulse width.

Diversity of Lactobacillus Species of Stilton Cheese Relates to Site of Isolation.

This study has characterized the dominant non-starter Lactobacillus species isolated from different sites in a Stilton cheese to establish its diversity, stress-tolerance, anti-microbial activity and potential contribution to quality of cheese. Fifty-nine Lactobacillus isolates were cultured from the outer crust, blue veins and white core of the cheese and were speciated phenotypically and by 16S rDNA sequence analysis. Lactobacillus plantarum was the dominant species detected with only two isolates identified as Lactobacillus brevis. Strains were typed by pulse-field gel electrophoresis (PFGE) using the enzyme NotI to examine their genomic diversity. Cluster analysis of PFGE patterns produced five major clusters which associated isolates with their sites of isolation within the cheese. One L. plantarum isolate from each cheese site was selected and evaluated for salt, acid, relative humidity, and heat tolerance to determine whether stress conditions within the isolation site selected their phenotype. D 72°C values were 6, 13, and 17 s for strains from the crust, veins and core, respectively, suggesting strains on the crust may not have been able to survive pasteurization and therefore had been added post-pasteurization. All strains recovered from heat injury within 24-48 h at 4°C. pH values of 3, 3.5, and 4 suppressed growth but strains showed a varying ability to grow at pH 4.5 and 5; isolates from the core (which has the lowest pH) were the most acid-tolerant. All strains grew at 3.5 and 5% salt but were suppressed at 10%; those from the crust (which has a lower water activity) were the most halo-tolerant, growing at 8% salt whereas strains from the core were sensitive to this salt concentration. All 57 L. plantarum isolates were examined for antimicrobial activity and variable activity against Lactobacillus pentosus and other genera was demonstrated; plantaricin EF genes were present in 65% of strains. It was concluded that there are varied phenotypes and genotypes of Lactobacillus in a Stilton cheese according to site of isolation. Occurrence of different L. plantarum genotypes could contribute to variation in the cheese quality from batch to batch and provides criteria for selecting isolates as potential adjunct cultures.