Probiotic interventions promote metabolic health in high fat-fed hamsters in association with gut microbiota and endocannabinoidome alterations.

Probiotics represent a promising tool to improve metabolic health, including lipid profiles and cholesterol levels. Modulation of the gut microbiome and the endocannabinoidome - two interrelated systems involved in several metabolic processes influenced by probiotics - has been proposed as a potential mechanism of action. This study establishes the impact of probiotics on metabolic health, gut microbiota composition and endocannabinoidome mediators in an animal model of hypercholesterolaemia. Syrian hamsters were fed either a low-fat low-cholesterol or high-fat high-cholesterol (HFHC) diet to induce hypercholesterolaemia and gavaged for 6 weeks with either Lactobacillus acidophilus CL1285, Lactiplantibacillus plantarum CHOL-200 or a combination of the two. Globally, probiotic interventions ameliorated, at least partially, lipid metabolism in HFHC-fed hamsters. The interventions, especially those including L. acidophilus, modified the gut microbiota composition of the small intestine and caecum in ways suggesting reversal of HFHC-induced dysbiosis. Several associations were observed between changes in gut microbiota composition and endocannabinoidome mediators following probiotic interventions and both systems were also associated with improved metabolic health parameters. For instance, potential connexions between the Eubacteriaceae and Deferribacteraceae families, levels of 2­palmitoylglycerol, 2­oleoylglycerol, 2­linoleoylglycerol or 2­eicosapentaenoylglycerol and improved lipid profiles were found. Altogether, our results suggest a potential crosstalk between gut microbiota and the endocannabinoidome in driving metabolic benefits associated with probiotics, especially those including L. acidophilus, in an animal model of hypercholesterolaemia.

Echocardiographic and electrocardiographic effects of atenolol versus sotalol in dogs with severe subaortic stenosis.

BACKGROUND: Subaortic stenosis (SAS) is a commonly diagnosed canine congenital cardiac defect, with severe forms of carrying a poor long-term prognosis. To date, an effective treatment strategy has not been developed in veterinary medicine. This study sought to determine if sotalol, a class III antiarrhythmic, may have salient echocardiographic and antiarrhythmic benefits for medical management for dogs affected with severe SAS. METHODS: Ten dogs diagnosed with severe SAS were enrolled in this prospective, double-blinded, crossover study. Dogs underwent physical exam, non-invasive blood pressure measurement, electrocardiography, echocardiography, and 24-h Holter monitoring. Diagnostics were repeated 12-16 days following randomization to oral atenolol (0.5-1 mg/kg) or sotalol (1-2 mg/kg) twice daily. After a medication taper and four-day washout, dogs were crossed-over to the alternate study medication, and the diagnostics were repeated in 12-16 days. Linear and multinomial mixed models were developed to evaluate the effects of treatments on echocardiographic and electrocardiographic variables. RESULTS: Indices of left ventricular systolic function were reduced based on the volumetric assessment when dogs received sotalol compared to atenolol. No difference was noted between groups in left ventricular systolic function based on the linear assessment. No difference was observed in the reduction in left ventricular outflow tract velocity. No significant differences were observed between treatment groups for any variable on 24-h Holter monitor. CONCLUSIONS: Sotalol may be a viable therapy to consider for dogs with severe SAS based on this pilot study. A larger, prospective study is necessary to investigate further.

Epicardial pacemaker implantation and outcome in eight pet ferrets (Mustela putorius furo).

INTRODUCTION/OBJECTIVES: Minimal information exists regarding epicardial pacemaker (EP) implantation in pet ferrets (Mustela putorius furo). The objectives were to describe the indications, surgical technique, and outcome of EP implantation in ferrets for the treatment of advanced atrioventricular block (AVB). ANIMALS, MATERIALS, AND METHODS: Eight client-owned ferrets presenting to five veterinary referral centers. Signalment, physical exam findings, diagnostic tests, anesthesia protocols, surgical implantation techniques, postoperative treatment plans, and EP interrogations were reviewed. Intra- and postoperative, minor and major, and EP-related complications were established. Descriptive statistics were performed to report complication rates. Survival analyses were performed. RESULTS: All ferrets had advanced AVB: 3/8 had high-grade second-degree and 5/8 had third-degree. The primary clinical signs were collapse and weakness. Seven EP were implanted via a transdiaphragmatic approach and one via a left intercostal thoracotomy. Intraoperative complications occurred in 2/8 ferrets, both major. One ferret with severe comorbidities died during general anesthesia. Postoperative pacemaker-related complications were minor: inappropriate sinus beat sensing in 2/8 and occasional muscle fasciculations in 1/8. Two ferrets were alive at the time of manuscript submission, at 10 and 21 months postoperatively. The overall median survival time was 24 months. CONCLUSIONS: Implantation of EP was performed successfully in most ferrets for treatment of advanced AVB and was well tolerated. Ferrets with advanced AVB may experience resolution of clinical signs associated with their cardiac disease following EP implantation. Additional studies are warranted to investigate the effects of epicardial pacing on survival times in this species.

Molecular mechanisms of activation and regulation of ANO1-Encoded Ca2+-Activated Cl- channels.

Ca2+-activated Cl- channels (CaCCs) perform a multitude of functions including the control of cell excitability, regulation of cell volume and ionic homeostasis, exocrine and endocrine secretion, fertilization, amplification of olfactory sensory function, and control of smooth muscle cell contractility. CaCCs are the translated products of two members (ANO1 and ANO2, also known as TMEM16A and TMEM16B) of the Anoctamin family of genes comprising ten paralogs. This review focuses on recent progress in understanding the molecular mechanisms involved in the regulation of ANO1 by cytoplasmic Ca2+, post-translational modifications, and how the channel protein interacts with membrane lipids and protein partners. After first reviewing the basic properties of native CaCCs, we then present a brief historical perspective highlighting controversies about their molecular identity in native cells. This is followed by a summary of the fundamental biophysical and structural properties of ANO1. We specifically address whether the channel is directly activated by internal Ca2+ or indirectly through the intervention of the Ca2+-binding protein Calmodulin (CaM), and the structural domains responsible for Ca2+- and voltage-dependent gating. We then review the regulation of ANO1 by internal ATP, Calmodulin-dependent protein kinase II-(CaMKII)-mediated phosphorylation and phosphatase activity, membrane lipids such as the phospholipid phosphatidyl-(4,5)-bisphosphate (PIP2), free fatty acids and cholesterol, and the cytoskeleton. The article ends with a survey of physical and functional interactions of ANO1 with other membrane proteins such as CLCA1/2, inositol trisphosphate and ryanodine receptors in the endoplasmic reticulum, several members of the TRP channel family, and the ancillary Κ+ channel ß subunits KCNE1/5.

Single left coronary ostium with a prepulmonic right coronary artery course in a French Bulldog with congenital valvular pulmonary stenosis.

A two-year-old, male castrated, French bulldog was presented for evaluation of coronary artery anatomy before balloon valvuloplasty for severe pulmonic valve stenosis. Multidetector computed tomography angiography showed a single left coronary ostium, absent right coronary ostium, and an anomalous, prepulmonic coursing right coronary artery. Medical management was elected to avoid the attendant risk associated with intervention. This case report documented the first known case of this specific anomaly in French bulldogs. Veterinary cardiologists should be aware of the potential for this specific coronary artery anomaly in this breed, given the predilection for the development of pulmonary stenosis. Routine screening of French bulldogs for anomalous, prepulmonic coronary arteries is recommended before interventional balloon valvuloplasty.

Evaluation of the NuCLEUS-X™ balloon valvuloplasty catheter for severe pulmonic stenosis in dogs.

INTRODUCTION/OBJECTIVES: Balloon instability is commonly encountered during balloon pulmonary valvuloplasty (BPV) and may result in an unsuccessful procedure. The NuCLEUS-X™ catheter is a recently developed BPV catheter with a unique barbell shape and an ordered pattern of inflation that stabilizes the balloon to span the valve annulus before expansion of the balloon center. ANIMALS: Ten client-owned dogs with severe valvular pulmonic stenosis (PS). MATERIALS AND METHODS: Prospective observational study. The BPV procedure was performed by standard technique with use of NuCLEUS-X™ catheters targeting a balloon-to-annulus ratio between 1.2 and 1.5. Balloon stability, safety, and procedural success were assessed. Procedural success was defined as either a reduction in the Doppler transpulmonic PG by at least 50% of the pre-procedural PG or <80 mmHg one month post procedure. RESULTS: Balloon stability centered at the pulmonic valve on the first inflation was achieved in 10/10 cases. The mean PG before BPV was 141 mmHg ±41 mmHg, and the PG after BPV at one month was 83 mmHg ±41 mmHg. Procedural success was achieved in 56% of patients. All dogs survived the BPV, and no major procedural complications were encountered using the NuCLEUS-X™ catheter. CONCLUSIONS: The use of the NuCLEUS-X™ catheter is feasible for BPV in dogs with severe PS. The unique balloon shape provided catheter stability on the first inflation in all dogs, which may be beneficial when stabilization of a conventional BPV catheter cannot be achieved.

Left atrial size and volume in cats with primary cardiomyopathy with and without congestive heart failure.

INTRODUCTION/OBJECTIVES: Myocardial diseases are the most common acquired cardiac diseases in cats and may result in left atrial enlargement and congestive heart failure (CHF). Volume calculations have replaced linear measurements for chamber quantification in humans but are not commonly measured in cats. The aims of this retrospective study were to compare the left atrial (LA) size by two-dimensional linear measurements to two-dimensional LA volumes (LAV). ANIMALS: One hundred sixty-two client-owned cats were included. MATERIALS AND METHODS: Cats with complete echocardiographic examinations were included and categorized into one of the three groups: healthy, cardiomyopathy (CM), and CHF. Seven measurements of the LA size were performed including minimal and maximal LA-to-aortic ratio (LA:Ao) and LAV and also maximal left atrial diameter (LAD). RESULTS: Cats were classified as healthy (n = 56), CM (n = 62), and CHF (n = 44). The minimal LA:Ao (LA:Aomin) and minimal LAV from the left apical view (LAVmin-LAP) best differentiated the CM and CHF groups. The LA:Aomin value with the optimal sensitivity and specificity to distinguish CM and CHF cats was 1.64 (sensitivity 84% and specificity 75%). CONCLUSIONS: Left atrial volumes were not superior to linear measurements of LA size in distinguishing CM and CHF cats in this study. Minimal LA size and volumes resulted in a larger area under the curve than each corresponding maximal value. Minimum LA size may be a better prognostic factor of CHF in cats with CM.

Inotropic and chronotropic effects of sotalol in healthy dogs.

INTRODUCTION: Sotalol is an anti-arrhythmic drug commonly used for the treatment of pathologic tachyarrhythmias in dogs. The ß-adrenergic blockade associated with sotalol administration may result in reduced myocardial contractility, which is clinically relevant for treating dogs with arrhythmias and concurrent myocardial dysfunction. The inotropic properties of sotalol are not well characterized in dogs. ANIMALS, MATERIALS, AND METHODS: Ten healthy, adult, large breed dogs were prospectively enrolled. All dogs underwent physical examination, blood pressure measurement, electrocardiography, 24-h Holter monitoring, and echocardiography including three-dimensional left ventricular volume measurements. Dogs were subsequently administered sotalol (1-2 mg/kg) orally twice daily for 12-16 days, and the same diagnostic tests were performed. Paired statistical analysis was used to compare parameters at baseline and after treatment with sotalol. RESULTS: Standard echocardiographic parameters of systolic function were reduced on sotalol compared to baseline, including ejection fraction via Simpson's method of disks which was 5.8% (95% confidence interval [CI]: 2.77-8.83%, p = 0.002) lower post-treatment. Maximum heart rate on Holter monitor was 17 bpm (95% CI: 9-37 bpm, p = 0.002) lower post-treatment than at baseline. CONCLUSIONS: Sotalol has a mild negative inotropic effect in healthy dogs based on standard echocardiographic measurements. There is also a negative chronotropic effect at higher heart rates based on 24-h Holter monitoring.

Prevalence of major complications and procedural mortality in 336 dogs undergoing interventional cardiology procedures in a single academic center.

INTRODUCTION: Overall complication rates associated with a wide range of diagnostic and therapeutic interventional cardiac procedures in a contemporary academic setting have not been reported. ANIMALS, MATERIALS AND METHODS: Consecutive interventional procedures performed for client-owned dogs were retrospectively analyzed to characterize procedural complications and mortality. RESULTS: Three hundred sixty-four procedures were performed on 336 dogs. Interventions included attempted or completed transvenous pacemaker (PM) implantation (n = 134) with subsequent pacing system revision (n = 8), pulmonic balloon valvuloplasty (BVP) (n = 117) with a subset of patients undergoing an additional BVP (n = 14), transarterial closure of left-to-right shunting patent ductus arteriosus (PDA) (n = 66), diagnostic angiography and/or cardiovascular pressure measurement (n = 9), transvenous temporary pacing (n = 7), septal defect occlusion (n = 5), heartworm extraction (n = 3), and BVP catheter fragment retrieval (n = 1). The prevalence of major perioperative and postoperative complications for all procedures was 5% and 6%, respectively, and the procedural mortality rate was 2%. The overall rate of major complications was 12% for the PM group, 11% for the BVP group, and 2% for the PDA occlusion group. Both PM implantation and BVP have higher rates of major complications overall compared with PDA occlusion (p=0.0151). CONCLUSIONS: The results of this study indicate that the prevalence of major complications and mortality associated with interventional cardiac procedures is low; however, significant differences exist in complication rates between procedures.

Interreader agreement of radiographic left atrial enlargement in dogs and comparison to echocardiographic left atrial assessment.

INTRODUCTION: Assessing left atrial (LA) size is an integral part of the cardiac evaluation in dogs. Left atrial size is routinely evaluated by thoracic radiographs or echocardiography. OBJECTIVES: The objectives of the study were to assess agreement of subjective radiographic left atrial enlargement (LAE) between readers, to compare subjective radiographic LAE with echocardiography, and to assess the accuracy and reliability of commonly used Roentgen signs for LAE. ANIMALS: One hundred one dogs with thoracic radiographs and echocardiography performed on the same day at a veterinary teaching hospital were retrospectively reviewed. METHODS: Thoracic radiographs were reviewed by two cardiologists, two radiologists, and two small animal rotating interns. Radiographs were evaluated for the subjective presence and severity of LAE and for seven Roentgen signs. Echocardiographic LA size was evaluated objectively by the left atrial-to-aortic root ratio and LA volume indexed to body weight. Interreader agreement of radiographic LAE evaluation and agreement between radiographic LAE assessment and echocardiographic LAE were assessed by linearly weighted kappa and intraclass correlation coefficient. RESULTS: Interreader agreement for the presence and degree of radiographic LAE ranged from moderate to substantial. The agreement between subjective radiographic LAE and echocardiographic LAE was moderate. Single Roentgen signs had poor to fair correlation with echocardiographic LAE. CONCLUSIONS: The present study found that interreader agreement for radiographic classification of LAE was substantial among specialists and moderate for all readers. Subjective, global assessment of radiographic LAE is better than using any single Roentgen sign.

Dielectric properties of isolated adrenal chromaffin cells determined by microfluidic impedance spectroscopy.

Knowledge of the dielectric properties of biological cells plays an important role in numerical models aimed at understanding how high intensity ultrashort nanosecond electric pulses affect the plasma membrane and the membranes of intracellular organelles. To this end, using electrical impedance spectroscopy, the dielectric properties of isolated, neuroendocrine adrenal chromaffin cells were obtained. Measured impedance data of the cell suspension, acquired between 1kHz and 20MHz, were fit into a combination of constant phase element and Cole-Cole models from which the effect of electrode polarization was extracted. The dielectric spectrum of each cell suspension was fit into a Maxwell-Wagner mixture model and the Clausius-Mossotti factor was obtained. Lastly, to extract the cellular dielectric parameters, the cell dielectric data were fit into a granular cell model representative of a chromaffin cell, which was based on the inclusion of secretory granules in the cytoplasm. Chromaffin cell parameters determined from this study were the cell and secretory granule membrane specific capacitance (1.22 and 7.10µF/cm2, respectively), the cytoplasmic conductivity, which excludes and includes the effect of intracellular membranous structures (1.14 and 0.49S/m, respectively), and the secretory granule milieu conductivity (0.35S/m). These measurements will be crucial for incorporating into numerical models aimed at understanding the differential poration effect of nanosecond electric pulses on chromaffin cell membranes.

Quantitative evaluation of left atrial volume and function by one-dimensional, two-dimensional, and three-dimensional echocardiography in a population of normal dogs.

OBJECTIVES: The primary goal of this study was to compare left atrial (LA) volume and functional indices in a population of normal dogs using transthoracic one-dimensional M-mode, two-dimensional (2D), and three-dimensional (3D) echocardiography. ANIMALS: Forty clinically normal dogs of various breeds were stratified into four groups based on weight. METHODS: Left atrial volume (LAV) was estimated by converting single linear measurements derived from M-mode and 2D images into volumes via the cube and sphere formulas. 2D echocardiography was employed to estimate LAV using linear measures and rectangular and ellipsoid formulas, as well as area-length and the monoplane method of disks. LAV was estimated using the biplane area-length method on 4- and 2-chamber 2D images. LAV was directly measured using 3D volume data obtained over four consecutive cardiac cycles. LAV estimates were compared amongst methods, correlated to body size, and evaluated for reproducibility. Functional indices derived from these various echocardiographic methods were compared to identify any systematic differences based on imaging modality. LA functional indices included LA ejection fraction (LAEF), total LA emptying volume, passive LA emptying volume/fraction, active LA emptying volume/fraction, and LA expansion index. RESULTS: The M-mode derived estimates of LAV were significantly smaller than all 2D- and 3D-derived LAV (p<0.0001). The left-sided 3D LAV had the highest correlation with body weight (r2 = 0.8806) for all phases of the LA cycle with acceptable interobserver variability (CV 11%). CONCLUSIONS: 3D echocardiography is a feasible, non-invasive method to measure LAV in a population of normal dogs.

Complete transposition of the great arteries with double outlet right ventricle in a dog.

A 2-year old intact male Collie dog presented to the cardiology service at Oregon State University for evaluation of cyanosis and suspected congenital cardiac disease. Echocardiography revealed a constellation of cardiac abnormalities including a single large vessel exiting the right ventricle with a diminutive left ventricular outflow tract, a ventricular septal defect, and marked concentric right ventricular hypertrophy with moderate right atrial dilation. Cardiac-gated computed tomography confirmed the previous anomalies in addition to supporting a diagnosis of complete transposition of the great arteries, double outlet right ventricle, and pulmonic hypoplasia with a single coronary ostium. Prominent bronchoesophageal collateral vessels were concurrently identified. Clinically, the dog was stable despite mild cyanosis that worsened with exercise; no intervention was elected at the time. This case report describes a rare combination of congenital cardiac defects and the usefulness of cardiac-gated cross-sectional imaging in the anatomic diagnosis.

Assessment of Preparation of Samples Under the Field Conditions and a Portable Real-Time RT-PCR Assay for the Rapid On-Site Detection of Newcastle Disease Virus.

Newcastle disease virus (NDV), also known as virulent forms of avian paramyxovirus serotype 1 (AMPV-1), is the causative agent of Newcastle disease affecting many species of birds and causing heavy losses to the poultry industry worldwide. Early, rapid and sensitive detection of the viruses or the viral nucleic acids is very important for disease diagnosis and control. This study aimed to evaluate sample preparation under field conditions and the application of a real-time RT-PCR method in the portable T-COR4 platform for the rapid, on-site detection of NDV on a farm. In the laboratory setting, the portable real-time RT-PCR assay had a similar performance compared with that obtained with a larger, stationary Rotor Gene real-time thermocycler. In the field conditions, viral nucleic acids were manually extracted just outside of animal units with minimal equipment and real-time RT-PCR detection was performed with the portable thermocycler T-COR4 placed in a nearby room. The portable assay at the farm detected viral RNA in 15 samples and reached an agreement of 83% (39/47) when the same RNA preparations were tested in the Rotor Gene thermocycler under the laboratory setting. The results demonstrated the feasibility of performing field detection but also the need to improve and further simplify sample preparation procedures.

Inhibitory role of phosphatidylinositol 4,5-bisphosphate on TMEM16A-encoded calcium-activated chloride channels in rat pulmonary artery.

BACKGROUND AND PURPOSE: Calcium-activated chloride channels (CaCCs) are key depolarizing mechanisms that have an important role in vascular smooth muscle contraction. Here, we investigated whether these channels are regulated by phosphatidylinositol (4,5) bisphosphate [P(4,5)P2 ], a known regulator of various ion channels. EXPERIMENTAL APPROACH: Calcium-activated Cl(-) currents (IClCa ) were recorded by patch clamp electrophysiology of rat isolated pulmonary artery smooth muscle cells. TMEM16A protein-phosphoinositide interaction was studied by co-immunoprecipitation and phosphoinositide binding arrays on protein lysates from whole pulmonary arteries and HEK293 cells overexpressing TMEM16A, the molecular correlate. KEY RESULTS: PI(4,5)P2 and other phospholipids were shown to bind directly to TMEM16A isolated from whole pulmonary artery (PA) and TMEM16A-eGFP expressed in HEK293 cells. Agents that reduced PI(4,5)P2 levels through different routes [PLC activation, PI4K inhibition, PI(4,5)P2 scavenging and absorption] all increased IClCa evoked by solutions containing clamped-free [Ca(2+) ], whereas enrichment of activating solutions with PI(4,5)P2 inhibited IClca in PA smooth muscle cells with approximately 50% reduction at 1 µM. CONCLUSIONS AND IMPLICATIONS: These data are the first to show a negative regulation of TMEM16A-encoded CaCCs by PI(4,5)P2 and propose that control of PI(4,5)P2 levels is a key determinant of arterial physiology.

Development of a suspension microarray for the genotyping of African swine fever virus targeting the SNPs in the C-terminal end of the p72 gene region of the genome.

African swine fever virus (ASFV) causes one of the most dreaded transboundary animal diseases (TADs) in Suidae. African swine fever (ASF) often causes high rates of morbidity and mortality, which can reach 100% in domestic swine. To date, serological diagnosis has the drawback of not being able to differentiate variants of this virus. Previous studies have identified the 22 genotypes based on sequence variation in the C-terminal region of the p72 gene, which has become the standard for categorizing ASFVs. This article describes a genotyping assay developed using a segment of PCR-amplified genomic DNA of approximately 450 bp, which encompasses the C-terminal end of the p72 gene. Complementary paired DNA probes of 15 or 17 bp in length, which are identical except for a single nucleotide polymorphism (SNP) in the central position, were designed to either individually or in combination differentiate between the 22 genotypes. The assay was developed using xMAP technology; probes were covalently linked to microspheres, hybridized to PCR product, labelled with a reporter and read in the Luminex 200 analyzer. Characterization of the sample was performed by comparing fluorescence of the paired SNP probes, that is, the probe with higher fluorescence in a complementary pair identified the SNP that a particular sample possessed. In the final assay, a total of 52 probes were employed, 24 SNP pairs and 4 for general detection. One or more samples from each of the 22 genotypes were tested. The assay was able to detect and distinguish all 22 genotypes. This novel assay provides a powerful novel tool for the simultaneous rapid diagnosis and genotypic differentiation of ASF.

Detection and phylogenetic analysis of peste des petits ruminants virus isolated from outbreaks in Punjab, Pakistan.

Peste des Petits Ruminants (PPR) is an important viral disease of small ruminants and is endemic in Pakistan. In the following study, samples from two outbreaks of PPR in goats have been subjected to laboratory investigations. The Peste des Petits Ruminants virus (PPRV) genome was detected using both conventional and real-time PCR. Genetic characterization of the local PPRV field isolates was conducted by sequencing 322 bp of the fusion (F) gene and 255 bp of the nucleoprotein (N) gene. The phylogenetic tree based on the F gene clustered samples from both outbreaks into lineage 4 along with other Asian isolates, specifically into subcluster 1 along with isolates from Middle East. Analysis of N gene revealed a different pattern. In this case, the Pakistani samples clustered with Chinese, Tajikistani and Iranian isolates, which probably represents the true geographical pattern of virus circulation. This is the first report presenting the phylogenetic tree based on N gene as well as performing a parallel comparison of the trees of F and N gene together from Pakistani isolates. The results of this study shed light on the PPRV population in Pakistan and emphasize the importance of using molecular methods to understand the epidemiology. Such understanding is essential in any efforts to control the number and impact of outbreaks that are occurring in endemic countries such as Pakistan, especially in the current scenario where OIE and FAO are eager to control and subsequently eradicate PPR from the globe, as has been achieved for Rinderpest.

Design and verification of a highly reliable Linear-After-The-Exponential PCR (LATE-PCR) assay for the detection of African swine fever virus.

African swine fever virus (ASFV) is a highly pathogenic DNA virus that is the causative agent of African swine fever (ASF), an infectious disease of domestic and wild pigs of all breeds and ages, causing a range of syndromes. Acute disease is characterized by high fever, haemorrhages in the reticuloendothelial system, and a high mortality rate. A powerful novel diagnostic assay based on the Linear-After-The-Exponential-PCR (LATE-PCR) principle was developed to detect ASFV. LATE-PCR is an advanced form of asymmetric PCR which results in direct amplification of large amount of single-stranded DNA. Fluorescent readings are acquired using endpoint analysis after PCR amplification. Amplification of the correct product is verified by melting curve analysis. The assay was designed to amplify the VP72 gene of ASFV genome. Nineteen ASFV DNA cell culture virus strains and three tissue samples (spleen, tonsil, and liver) from infected experimental pigs were tested. Virus was detected in all of the cell culture and tissue samples. None of five ASFV-related viruses tested produced a positive signal, demonstrating the high specificity of the assay. The sensitivity of the LATE-PCR assay was determined in two separate real-time monoplex reactions using samples of synthetic ASFV and synthetic control-DNA targets that were diluted serially from 109 to 1 initial copies per reaction. The detection limit was 1 and 10 copies/reaction, respectively. The sensitivity of the assay was also tested in a duplex end-point reactions comprised of a constant level of 150 copies of synthetic control-DNA and a clinical sample of spleen tissue diluted serially from 10⁻¹ to 10⁻5. The detection limit was 10⁻5 dilution which corresponds to approximately 1 copy/reaction. Since the assay is designed to be used in either laboratory settings or in a portable PCR machine (Bio-Seeq Portable Veterinary Diagnostics Laboratory; Smiths Detection, Watford UK), the LATE-PCR provides a robust and novel tool for the diagnosis of ASF both in the laboratory and in the field.

Development of a real-time RT-PCR assay based on primer-probe energy transfer for the detection of all serotypes of bluetongue virus.

A real-time RT-PCR assay based on the primer-probe energy transfer (PriProET) was developed to detect all 24 serotypes of bluetongue virus (BTV). BTV causes serious disease, primarily in sheep, but in other ruminants as well. A distinguishing characteristic of the assay is its tolerance toward mutations in the probe region. Furthermore, melting curve analysis following immediately PCR confirms specific probe hybridization and can reveal mutations in the probe region by showing a difference in the melting point. The assay sensitivity was in the range of 10-100 target copies and the specificity tests showed no positive results for heterologous pathogens. The assay was tested on clinical samples from BTV 8 outbreaks in Sweden and Denmark in 2008. The lowest detection limit for that serotype, determined with PCR standards, was 57 genome copies. The assay sensitivity for some other serotypes that circulate currently in Europe was also determined. BTV 2, 4, 9 and 16 were tested on available cell culture samples and the detection limits were 109, 12, 13 and 24 copies, respectively. This assay provides an important tool for early and rapid detection of a wide range of BTV strains, including emerging strains.

Phosphorylation alters the pharmacology of Ca(2+)-activated Cl channels in rabbit pulmonary arterial smooth muscle cells.

BACKGROUND AND PURPOSE: Ca(2+)-activated Cl(-) currents (I(Cl(Ca))) in arterial smooth muscle cells are inhibited by phosphorylation. The Ca(2+)-activated Cl(-) channel (Cl(Ca)) blocker niflumic acid (NFA) produces a paradoxical dual effect on I(Cl(Ca)), causing stimulation or inhibition at potentials below or above 0 mV respectively. We tested whether the effects of NFA on I(Cl(Ca)) were modulated by phosphorylation. EXPERIMENTAL APPROACH: I(Cl(Ca)) was elicited with 500 nM free internal Ca(2+) in rabbit pulmonary artery myocytes. The state of global phosphorylation was altered by cell dialysis with either 5 mM ATP or 0 mM ATP with or without an inhibitor of calmodulin-dependent protein kinase type II, KN-93 (10 microM). KEY RESULTS: Dephosphorylation enhanced the ability of 100 microM NFA to inhibit I(Cl(Ca)). This effect was attributed to a large negative shift in the voltage-dependence of block, which was converted to stimulation at potentials <-50 mV, approximately 70 mV more negative than cells dialysed with 5 mM ATP. NFA dose-dependently blocked I(Cl(Ca)) in the range of 0.1-250 microM in cells dialysed with 0 mM ATP and KN-93, which contrasted with the stimulation induced by 0.1 microM, which converted to block at concentrations >1 microM when cells were dialysed with 5 mM ATP. CONCLUSIONS AND IMPLICATIONS: Our data indicate that the presumed state of phosphorylation of the pore-forming or regulatory subunit of Cl(Ca) channels influenced the interaction of NFA in a manner that obstructs interaction of the drug with an inhibitory binding site.