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Aims Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by ventricular dysfunction and ventricular arrhythmias (VA). Adequate arrhythmic risk assessment is important to prevent sudden cardiac death. We aimed to study the incremental value of strain by feature-tracking cardiac magnetic resonance imaging (FT-CMR) in predicting sustained VA in ARVC patients. Methods and results CMR images of 132 ARVC patients (43% male, 40.6 ± 16.0 years) without prior VA were analysed for global and regional right and left ventricular (RV, LV) strain. Primary outcome was sustained VA during follow-up. We performed multivariable regression assessing strain, in combination with (i) RV ejection fraction (EF); (ii) LVEF; and (iii) the ARVC risk calculator. False discovery rate adjusted P-values were given to correct for multiple comparisons and c-statistics were calculated for each model. During 4.3 (2.0–7.9) years of follow-up, 19% of patients experienced sustained VA. Compared to patients without VA, those with VA had significantly reduced RV longitudinal (P ≤ 0.03) and LV circumferential (P ≤ 0.04) strain. In addition, patients with VA had significantly reduced biventricular EF (P ≤ 0.02). After correcting for RVEF, LVEF, and the ARVC risk calculator separately in multivariable analysis, both RV and LV strain lost their significance [hazard ratio 1.03–1.18, P > 0.05]. Likewise, while strain improved the c-statistic in combination with RVEF, LVEF, and the ARVC risk calculator separately, this did not reach statistical significance (P ≥ 0.18). Conclusion Both RV longitudinal and LV circumferential strain are reduced in ARVC patients with sustained VA during follow-up. However, strain does not have incremental value over RVEF, LVEF, and the ARVC VA risk calculator.

Background The Task Force Criteria (TFC) for ARVC are highly sensitive, but lack specificity. Patients with atypical RV-involvement (aRVi) may have different underlying aetiologies and prognosis, requiring specific therapeutic interventions. Purpose We aimed to evaluate the role of the baseline ECG for initial suspicion of aRVi. Methods From the Netherlands Heart Institute Arrhythmogenic Cardiomyopathy (NHI-ACM) registry, patients were selected who 1) fulfilled TFC for definite ARVC, 2) presented with sustained VT, 3) underwent genetic testing. The first available ECG after VT was evaluated for AV-conduction and the presence and surface area (SA) of an R'-wave in V1-V3. ECGs with AV-conduction disturbances or an R'-wave with SA ≥1.65 mm2 were classified as suspicious for `atypical RV-involvement' (aRVi-ECG). Patients with ARVC-related pathogenic/likely pathogenic variant (P/LP+) were classified as “typical ARVC”. Data of patients without an ARVC-related pathogenic/likely pathogenic variant (P/LP−) were reviewed by an expert panel and classified as either “typical ARVC” or “suggestive for another aetiology” based on consensus. Results In total 124 P/LP+ patients and 35 P/LP− patients were included. Nineteen patients had an aRVi-ECG, which appeared significantly more predominant in the P/LP− group (11 (9%) P/LP+ vs. 8 (22%) P/LP−, p=0.019). Of the P/LP− patients, seventeen (49%) were classified as “suggestive for another aetiology” (e.g. myocarditis, ischemia, sarcoid), including all 8 patients with an aRVI-ECG. Among P/LP+ patients with an aRVi-ECG, 46% carried the Arg14del phospolamban variant and 64% died, versus 15% and 18% of P/LP+ patients without aRVi-ECG, respectively (Table 1). Conclusion For patients presenting with sustained VT and fulfilling the TFC for ARVC diagnosis, a baseline ECG suggestive for atypical RV-involvement should raise suspicion for a different underlying aetiology in patients without an ARVC-related P/LP variant. In P/LP+ patients, an aRVi-ECG may identify those with poor outcome. Funding Acknowledgement Type of funding sources: None.

Background A pathogenic variant in the gene encoding phospholamban (PLN; p.Arg14del) may cause cardiomyopathy, the pathophysiologic hallmark of which is cardiac fibrosis that may cause malignant ventricular arrhythmias and severe heart failure. There is no evidence-based therapy to modify disease progression in presymptomatic pathogenic variant carriers. Eplerenone is a selective mineralocorticoid receptor antagonist with established antifibrotic properties, and may be a potentially interesting treatment in PLN p.Arg14del mutation carriers. Purpose To investigate if preventive therapy with eplerenone attenuates disease onset and progression in PLN cardiomyopathy. Methods 84 presymptomatic PLN p.Arg14del mutation carriers (median age 39 [27–50] years, 44% males) were randomized 1:1 to receive eplerenone (50mg once daily) or no treatment, and endpoints were adjudicated in a blinded fashion (PROBE design). Baseline values were acquired including demographics, cardiac magnetic resonance imaging (CMR) parameters (ventricular volumes, systolic function, late gadolinium enhancement (LGE)), and electrocardiographic parameters (QRS voltages and ventricular ectopy per 24 hour). Subjects were re-evaluated every year and CMR was performed at baseline and after three years. The composite endpoint included LVEDV/RVEDV increase (>10%), LVEF/RVEF decrease (5%), newly developed LGE on CMR, increase in ventricular ectopy (>100% PVCs/24h if >1000 PVCs/24h), decrease in QRS voltage (>25%), non-sustained ventricular tachycardia, symptoms or arrhythmias necessitating treatment, and cardiovascular death. Results Two out of 84 subjects (2%) were lost to follow up. 49 out of 82 remaining subjects (60%) reached the composite end point, 25 (60%) in the control group, 24 (60%) in the treatment group (p=0.825). There was no significant difference between the groups when considering the individual end points, see Table 1. Baseline and follow up CMR was performed on 76 subjects, 39 in the control group, 37 in the treatment group. In total there were 10 (13%) cases of newly developed LGE, 6 (18%) in the control group, 4 (12%) in the treatment group (p=0.556), see Figure 1. Conclusions Eplerenone did not significantly attenuate disease onset and progression in presymptomatic PLN p.Arg14del mutation carriers. There was no significant difference in new incidence of LGE between the treatment and control groups. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This work was financially supported by the Netherlands Cardiovascular Research Initiative, an initiative supported by the Dutch Heart Foundation (The Hague, Netherlands).

Background The p.Arg14del (c.40_42delAGA) phospholamban (PLN) pathogenic variant is a founder mutation that causes dilated cardiomyopathy (DCM) and arrhythmogenic cardiomyopathy (ACM). Carriers are at increased risk of malignant ventricular arrhythmias and heart failure, which has been ascribed to cardiac fibrosis. Importantly, cardiac fibrosis appears to be an early feature of the disease, occurring in many presymptomatic carriers before the onset of overt disease. As with most monogenic cardiomyopathies, no evidence-based treatment is available for presymptomatic carriers. Aims The PHOspholamban RElated CArdiomyopathy intervention STudy (iPHORECAST) is designed to demonstrate that pre-emptive treatment of presymptomatic PLN p.Arg14del carriers using eplerenone, a mineralocorticoid receptor antagonist with established antifibrotic effects, can reduce disease progression and postpone the onset of overt disease. Methods iPHORECAST has a multicentre, prospective, randomised, open-label, blinded endpoint (PROBE) design. Presymptomatic PLN p.Arg14del carriers are randomised to receive either 50 mg eplerenone once daily or no treatment. The primary endpoint of the study is a multiparametric assessment of disease progression including cardiac magnetic resonance parameters (left and right ventricular volumes, systolic function and fibrosis), electrocardiographic parameters (QRS voltage, ventricular ectopy), signs and/or symptoms related to DCM and ACM, and cardiovascular death. The follow-up duration is set at 3 years. Baseline results A total of 84 presymptomatic PLN p.Arg14del carriers (n = 42 per group) were included. By design, at baseline, all participants were in New York Heart Association (NHYA) class I and had a left ventricular ejection fraction > 45% and Conclusion iPHORECAST is a multicentre, prospective randomised controlled trial designed to address whether pre-emptive treatment of PLN p.Arg14del carriers with eplerenone can prevent or delay the onset of cardiomyopathy. iPHORECAST has been registered in the clinicaltrials.gov-register (number: NCT01857856).

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This work was supported by the Dutch Heart Foundation Introduction Non-invasive mapping of ventricular activation using inverse electrocardiography (iECG) in patients with cardiomyopathy during sinus rhythm, may improve risk stratification for sudden cardiac death. However, iECG is complicated by multiple simultaneous endocardial activation waves (multi-wave) mediated by the His-Purkinje system, especially when the QRS complex is narrow. The activation estimation should be based on a realistic physiological model of the His-Purkinje system combining multiple waves initiated at His-Purkinje associated endocardial locations. Equivalent double layer based iECG provides an estimation of both the endocardial and epicardial surface. To improve accuracy, equivalent double layer based iECG was supplemented with electro-anatomical structures associated with the His-Purkinje system to test initial ventricular activation (Figure, Panel C). Multi-wave iECG local activation timing (LAT) maps and invasive LAT maps during sinus rhythm were quantitatively compared. Purpose Quantitative comparison of multi-wave iECG in His-Purkinje mediated cardiac activation using invasive activation maps in patients. Methods Thirteen patients referred for invasive electro-anatomical mapping (EAM) of the endocardial and epicardial surface were included. Prior to EAM, each subject underwent 64 electrode body surface potential mapping, cardiac computed tomography (CT) imaging, and 3D imaging of electrode positions. Anatomical models of the ventricles, lungs and thorax were created using CT images and supplemented with electrode positions (Figure, Panel A-B). Electro-anatomical structures associated with the His-Purkinje system were incorporated in ventricular anatomical models (Figure, Panel C) and multiple simultaneous activation waves were simulated. Invasive endocardial and epicardial LAT maps were quantitatively compared to iECG LAT maps. Invasive EAM LAT maps were quantitatively compared to estimated iECG LAT maps (Figure, Panel D) using inter-map correlation coefficients (CC, Pearson’s) and absolute differences (AD). Results Mean inter-map CC and AD were 0.54 ± 0.19 and 18 ± 7 ms respectively for the epicardial surface (n = 13). Similar to the RV endocardial surface (n = 10, CC = 0.50 ± 0.29, AD = 20 ± 8 ms) and the LV endocardial surface (n = 4, CC = 0.44 ± 0.26, AD = 25 ± 7 ms). Conclusion(s): Quantitative comparison of the multi-wave iECG method showed overall moderate performance. This novel iECG method provides a physiologically more realistic and more robust estimation of sinus rhythm and may serve as a tool for detection of electro-anatomical substrates and risk stratification. Compared to other available non-invasive ECG methods, multi-wave iECG captures His-Purkinje mediated ventricular activation better. This method might also be useful for the accurate detection and localization of structural conduction disorders. Abstract Figure. Multi-Wave inverse electrocardiography

Background Noninvasive electrocardiographic imaging (ECGi) using the equivalent double layer (EDL) source model enables both epicardial and endocardial reconstruction of electroanatomical activation patterns during sinus rhythm. The EDL source model has been validated in torso-tank models and animal experiments, however validation in humans using invasive electroanatomical mapping is limited. Purpose Validation of EDL based ECGi using invasive electroanatomical mapping. Methods Ten patients referred for epicardial and endocardial electroanatomical mapping underwent 67 electrode body surface potential mapping (BSPM), cardiac CT imaging and 3D imaging of electrode positions. Anatomical models of the ventricles, lungs and thorax were created and supplemented with electrode positions. Invasive epicardial (4020±1514 contact points), right ventricle endocardial (724±113 contact points) and left ventricle endocardial (459±117 contact points) local activation timing (LAT) maps were compared to ECGi derived LAT maps. Results Included patients (mean age 48±21 years, 80% males) were diagnosed with either arrhythmogenic cardiomyopathy (N=4), dilated cardiomyopathy (N=1), symptomatic premature ventricular complexes (N=3) or myocarditis (N=2). Measured BSMP highly correlated with simulated BSPM (97±2%) with a relative difference of 24% ± 6%. Source models consisted of a mean of 2614±214 nodes with a mean distance between nodes of 8.3±1 mm. Overall, invasive LAT maps and ECGi derived LAT maps showed reasonable correlation for epicardial maps (49% ± 18%) and endocardial maps of the right ventricle (48% ± 27%). Endocardial left ventricular LAT maps showed less correlation (23% ± 28). The absolute difference between invasive and ECGi LAT maps was 16.7±14.2 ms for epicardial maps, 17.2±12.9 ms for right ventricle endocardial maps and 34.0±19.8 ms for left ventricle endocardial maps. Visual comparison showed corresponding areas of breakthrough and regions of latest activation in all cases (Figure). Quantitative comparison of these areas showed a mean absolute difference of 33±7mm or 19±10 ms for epicardial maps, 25±10 mm or 17±13 ms for right ventricle endocardial maps and 39±16 mm or 20±6 ms for left ventricular endocardial maps. Conclusion Overall agreement was observed between EDL based ECGi LAT maps and electroanatomical LAT maps. Future research will focus on further quantification of this agreement and improvement of ECGi mapping accuracy. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Netherlands Cardiovascular Research Initiative, an initiative with support of the Dutch Heart Foundation and UCL Hospitals NIHR Biomedical Research Centre.

Arrhythmogenic cardiomyopathy (AC), also known as arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C), is a hereditary disease characterised by ventricular arrhythmias, right ventricular and/or left ventricular dysfunction, and fibrofatty replacement of cardiomyocytes. Patients with AC typically present between the second and the fourth decade of life with ventricular tachycardias. However, sudden cardiac death (SCD) may be the first manifestation, often at young age in the concealed stage of disease. AC is diagnosed by a set of clinically applicable criteria defined by an international Task Force. The current Task Force Criteria are the essential standard for a correct diagnosis in individuals suspected of AC. The genetic substrate for AC is predominantly identified in genes encoding desmosomal proteins. In a minority of patients a non-desmosomal mutation predisposes to the phenotype. Risk stratification in AC is imperfect at present. Genotype-phenotype correlation analysis may provide more insight into risk profiles of index patients and family members. In addition to symptomatic treatment, prevention of SCD is the most important therapeutic goal in AC. Therapeutic options in symptomatic patients include antiarrhythmic drugs, catheter ablation, and ICD implantation. Furthermore, patients with AC and also all pathogenic mutation carriers should be advised against practising competitive and endurance sports.

Pathogenic mutations in the phospholamban (PLN) gene may give rise to inherited cardiomyopathies due to its role in calcium homeostasis. Several PLN mutations have been identified, with the R14del mutation being the most prevalent cardiomyopathy-related mutation in the Netherlands. It is present in patients diagnosed with arrhythmogenic cardiomyopathy as well as dilated cardiomyopathy. Awareness of the phenotype of this PLN mutation is of great importance, since many carriers remain to be identified. Patients with the R14del mutation are characterised by older age at onset, low-voltage electrocardiograms and ahigh frequency of ventricular arrhythmias. Additionally, these patients have apoor prognosis often with left ventricular dysfunction and early-onset heart failure. Therefore, when there is asuspicion of aPLN mutation, cardiac and genetic screening is strongly recommended.

In this study, 117 isolates of Haemophilus parasuis from organs and tissues from pigs showing clinical signs, were characterised and compared with 10 H. parasuis reference strains. The isolates were subjected to the 16S rRNA gene PCR and subsequently serotyped, genotyped by 60-kDa heat shock protein (Hsp60) gene sequences, the enterobacterial repetitive intergenic consensus (ERIC) PCR and a multiplex PCR for the detection of the vtaA virulence associated trimeric autotransporter genes. Serotyping revealed the presence of 13 H. parasuis serovars. Serovars 3 and 10 were not detected, and 16 of the 117 H. parasuis isolates could not be typed by specific antisera. All isolates were positive in the 16S rRNA gene specific H. parasuis PCR. ERIC-PCR revealed a very heterogeneous pattern with 61 clusters; based on a 90% agreement. In total, 46 different Hsp60 sequence types were detected. Using 98% sequence similarity, as threshold for separation, 22 separate Hsp60 sequence clusters were distinguished. There was no correlation between H. parasuis serovars and ERIC-PCR clusters or Hsp60 sequence types, but both the ERIC-PCR and the Hsp60 sequence typing are suited as markers for H. parasuis molecular-epidemiology studies. In total, 102 H. parasuis swine isolates corresponded to the virulence associated group 1 vtaA type. The group 1 vtaA was detected in 12 different serovars. Only four of the 46 Hsp60 sequence types were not associated with the group 1 vtaA. This study shows that Dutch H. parasuis isolates from pigs with clinical signs have both a high serovar and genotypic lineage diversity. A majority of the known serovars contain the group 1 vtaA.

In 2013, seven outbreaks of granuloma disease occurred in Dutch flocks of productive layers housed on different farms. These outbreaks were characterized by increased mortality and high incidence of granulomas, mainly in caeca (340/408 hens = 83%) and livers (69/408 hens = 17%). Mortality started to increase between 21 and 35 weeks of age and reached 3.7% to 11.0% exceeding the breeder's norm in periods ranging from 9 to 48 weeks. Some flocks also showed decreased egg production and/or loss of mean egg weight. All affected flocks were linked to one rearing farm, which therefore seemed to be the source of the disease. However, no signs of disease had been observed at this rearing farm. Sentinel hens placed in one of the affected flocks to determine whether the disease had an infectious nature developed granulomas identical to those seen in the outbreaks. Next, by fulfilling Koch's postulates it was shown that Tetratrichomonas gallinarum was the aetiological agent of the granuloma disease. The condition was reproduced in mature specified pathogen free White Leghorn hens (GD - Animal Health, Deventer, the Netherlands) by inoculation via both an artificial and a natural route with a well-defined axenic T. gallinarum isolate obtained from one of the affected flocks. Other causes of granuloma disease were excluded.

The history, current situation and control measures for infectious bronchitis virus (IBV) variants are reviewed. A large number of IBV variants exist worldwide; some being unique to a particular area, others having a more general distribution. The possible reasons why some strains spread readily over major parts of the world, whereas other strains stay more localized are discussed. The advantages and disadvantages of strain classification by protectotyping, serotyping and genotyping are discussed in relation to in vivo protection. The different vaccination strategies are also considered.

Histomonosis is an infectious disease of mainly galliform birds that can cause high mortality, especially in commercial turkey flocks. Several diagnostic tools were available to detect its causative agent, the flagellated protozoan Histomonas meleagridis. However, serological tools were largely lacking. Recently, an indirect ELISA has been described, but this test was not validated for specificity and might suffer from cross-reactivity with related protozoa. Therefore, a specific blocking-ELISA for the detection of antibodies against H. meleagridis in chicken and turkey sera was developed. For this purpose, monoclonal antibodies were raised against a detergent extracted protein of H. meleagridis. These MAbs bound to morphologically identified histomonads in liver tissue of an infected turkey. The MAbs were conjugated with horseradish peroxidase, after which the most reactive conjugate was used to set-up the blocking-ELISA. Experimentally infected turkeys (n=9) and chickens (n=10) seroconverted in the blocking-ELISA within 2-4 weeks following inoculation with a H. meleagridis field strain. The MAb did not bind to Tetratrichomonas gallinarum antigen and experimentally inoculated T. gallinarum seropositive layer chickens (n=18) showed the same inhibition percentages in the blocking-ELISA as negative control birds did. Therefore, it was concluded that H. meleagridis blocking-ELISA does not cross-react with T. gallinarum, which is a closely related protozoan frequently occurring in poultry. The repeatability and reproducibility of the H. meleagridis blocking-ELISA were high. The new blocking-ELISA is a promising tool for experimental studies; however, further validation of this test with field samples is necessary before it can be used for diagnostic purposes.

Background Over the past decade, radiofrequency catheter ablation (RFCA) of atrial fibrillation (AF) has evolved into a frequently performed procedure. The aim of this study was to monitor changes in patient characteristics, procedural characteristics, outcomes and complications over the past 10 years. Methods All consecutive patients who underwent primary RFCA treatment of AF in the University Medical Center Utrecht from 2005–2015 were included. In all patients, the primary ablation strategy was pulmonary vein (PV) antrum isolation without additional substrate modification. Baseline patient and procedure characteristics, and 1-year follow-up data of 975 patients were prospectively collected. Results In 2005, 73.4% of patients suffered from paroxysmal AF, which decreased to 45.3% in 2014. Mean age increased from 54 ± 9 to 61 ± 10 years and CHA2DS2-VASc score ≥2 from 18 to 40.6%. History of AF decreased significantly from 7 to 4 years. Mean procedure duration was 237 ± 53 min in 2005 and 163 ± 41 min in 2014. Fluoroscopy time significantly decreased from 41 ± 17 to 19 ± 8 min and total radiation exposure from 465 (263–687) to 210 (118–376) mGy. One-year success remained similar (2005: 55.6%, 2014: 54.8%), as did the amount of PV reconnection observed during redo procedures. Due to a marked reduction in vascular complications and moderate PV stenosis, the total complication rate decreased significantly. Conclusion Over the past decade, AF ablation has increasingly been performed in older patients with persistent AF and more comorbidity. Moreover, it has been performed earlier after AF diagnosis. Although several performance parameters, such as procedure duration and complication rate, improved, 1-year single procedure success remained unchanged.

Summary. Background and objectives : The key complication of treatment with vitamin K antagonists (VKAs) is bleeding. The major determinant of VKA-induced bleeding is the intensity of anticoagulation. Individual patient characteristics may also influence bleeding risk. In addition, soluble thrombomodulin (s-TM) levels and mutations in the propeptide of factor (F)IX are important candidate risk factors in this respect. Patients and methods : A matched case–control study was designed to search for risk factors that predict bleeding during VKA treatment. We selected cases that had experienced major bleeding during treatment with VKA and matched controls without bleeding complications from the databases of two Thrombosis Services. The controls were matched for indication of treatment, age, gender, type of anticoagulant used and whether or not treatment with VKA was stopped. DNA and plasma were stored of all cases and controls. Results and conclusions : In total 110 patients and 220 controls consented to participate. The results indicate that s-TM levels, measured by ELISA, may be a risk indicator for bleeding [crude odds ratio 3.25 for the highest quartile vs. the lowest quartile (95% confidence interval 1.40, 7.51)]. Three novel mutations, determined by direct sequencing, in the gene portion encoding the propeptide of FIX were identified that do not seem to play an important role in bleeding risk during treatment with VKAs.

Dwyer medium is the most frequently employed culture medium for Histomonas meleagridis. Both for subculturing and for resuscitation of H. meleagridis from storage in liquid nitrogen, modified Dwyer medium with an increased rice powder concentration (0.8%) and no chicken embryo extract proved superior to Dwyer standard medium, with threefold (10(6.3) vs. 10(5.8) histomonads/ml) to 10-fold (10(6.7) vs. 10(5.8) histomonads/ml) higher concentrations of parasites after resuscitation or subculturing, respectively.

Receiver operating characteristic (ROC) curves assess the quality of tests over the entire range of test signals. We compared the ability of an ELISA to detect antibodies to Aujeszky's disease (pseudorabies) virus gE in colostrum (test A) and in a single droplet of whole blood (test B) with the results obtained in serum (gold standard) in the target population by constructing and analyzing such curves. The area under the ROC curve, which is a quantitative measure of test performance, proved to be significantly (p < 0.01) smaller in test A than in test B or the gold standard. No significant differences in the area under the ROC curve were observed between test B and the gold standard.

Histomonosis, a parasitic disease of galliformes and sporadically of other birds caused by Histomonas meleagridis, can result in very high mortality, especially in turkeys. The ban on the last antihistomonal drug prompted an urgent search for alternative prevention and treatment strategies. As both paromomycin and tiamulin have been reported to have antihistomonal activity, these antibiotics were investigated in vitro by adding two-fold serial dilutions ranging from 12.5 to 400 microg/mL to cultures of H. meleagridis. Controls (no antibiotics, or 12.5 microg or 400 microg/mL dimetridazole) were included. Parasites were counted after 3, 20, 28, 44, 51, and 71 hours of incubation. Tiamulin did not have a clear antihistomonal effect, but paromomycin had an inhibitory effect at all concentrations tested. The latter antibiotic was subsequently examined in an in vivo study. Five groups of 20 1-day-old poults, matched by weight and sex, were either not treated (infected and uninfected control groups) or treated with paromomycin (100, 200, or 400 ppm) added to their feed. After 2 weeks all groups, except for the uninfected control group, were intracloacally inoculated with 200,000 histomonads per bird. A clear dose-response effect was found for paromomycin. In the 100-ppm paromomycin group, mortality was similar to that in the untreated control group, whereas about half of the birds died in the 200-ppm paromomycin group; almost complete protection against histomonosis was seen in the 400-ppm paromomycin group. This study shows that paromomycin supplied in feed at 400 ppm is a potentially preventive strategy against H. meleagridis.

Histomonas meleagridis is the etiological agent of histomonosis or blackhead disease. Recently, genotyping, based on polymerase chain reaction and sequencing of internal transcribed spacer-1 sequences was applied to various isolates originating from fowl. Three genotypes were described: types I and II isolates were associated with clinical disease and probably derived from H. meleagridis, whereas, type III isolates were not disease-associated and likely corresponded to Parahistomonas wenrichi according to morphological observations. However, this latter species has never been characterized at the molecular level and its phylogenetic relationships with other parabasalids remained hypothetical. To confirm the identification of these isolates, small subunit rRNA gene sequences were obtained from representatives of types I, II, and III and analyzed in a broad phylogeny including 64 other parabasalid sequences. From our phylogenetic trees, we confirmed that types I and II isolates were closely related, if not identical, to H. meleagridis, while type III isolates represented P. wenrichi. Both species clustered together with high support. This grouping suggested that speciation leading to these two species inhabiting the same hosts and ecological niche occurred recently in birds. In addition, speciation was likely followed by loss of pathogenicity in P. wenrichi.

Histomoniasis is a serious disease in poultry. All chemotherapeutics with known efficacy against its causative agent, Histomonas meleagridis, have been banned from use as prophylactic or therapeutic use in production animals. In a search for possible alternatives, the in vivo effects of the herbal products Enteroguard and Protophyt were examined. Two-week-old turkeys allocated into 13 groups of 18 birds were either sham inoculated (negative control group) or were inoculated with 100, 3162 or 200 000 histomonads per bird. Control groups (no feed additives, dimetridazole, or Histostat-50) were included in the study. No morbidity or mortality was observed in the negative control group or in the groups inoculated with 100 histomonads per bird. Mortality was 100% in the groups inoculated with 200 000 histomonads per bird and either untreated (positive control group) or receiving Protophyt SP, Protophyt SP and Protophyt B, Enteroguard, or Histostat-50. Mortality was 17% in the dimetridazole-treated group. In the groups inoculated with 3162 histomonads per bird, mortality was 100% for the positive control group and the group receiving Enteroguard, and was 94% in the group receiving Protophyt SP. In the present study, Enteroguard or Protophyt was not found to be effective against histomoniasis.

Dientamoeba fragilis is a parasite that has been recognized to be a causative agent of gastrointestinal symptoms. Because in most studies only some infected persons experience symptoms, it is possible that D. fragilis is a heterogeneous species with variants that display similar morphologies but different pathogenicities. The search for genetic variation in D. fragilis was based on the small-subunit rRNA gene, which was not found to be useful for molecular epidemiology. In this report, we describe the isolation and characterization of additional rRNA gene cluster sequences, the internal transcribed spacer 1 (ITS-1)-5.8S rRNA gene-ITS-2 region. For comparative purposes, we also isolated the ITS-1-5.8S rRNA gene-ITS-2 region of Histomonas meleagridis , a protozoan parasite of birds and a close relative of D. fragilis . This region was found to be highly variable, and 11 different alleles of the ITS-1 sequence could be identified. Variation in the ITS-1 region was found to be intragenomic, with up to four different alleles in a single isolate. So-called C profiles were produced from the ITS-1 repertoire of single isolates,. Analysis of the C profiles of isolates from nonrelated patients identified several clearly distinguishable strains of D. fragilis . Within families, it was shown that members can be infected with the same or different strains of D. fragilis . In conclusion, the ITS-1 region can serve as a molecular epidemiological tool for the subtyping of D. fragilis directly from feces. This may serve as a means of studying the transmission, geographical distribution, and relationships between strains and the pathogenicity of this parasite.

Cecal samples from laying chickens from 25 farms with a history of decreased egg production, diarrhea, and/or increased feed conversion ratios were examined for anaerobic intestinal spirochetes of the genus Brachyspira . Seventy-three samples positive in an immunofluorescence assay for Brachyspira species were further examined using selective anaerobic culture, followed by phenotypic analysis, species-specific PCRs (for Brachyspira hyodysenteriae , B. intermedia , and B. pilosicoli ), and a Brachyspira genus-specific PCR with sequencing of the partial 16S rRNA gene products. Brachyspira cultures were obtained from all samples. Less than half of the isolates could be identified to the species level on the basis of their biochemical phenotypes, while all but four isolates (5.2%) were speciated by using PCR and sequencing of DNA extracted from the bacteria. Different Brachyspira spp. were found within a single flock and also in cultures from single chickens, emphasizing the need to obtain multiple samples when investigating outbreaks of avian intestinal spirochetosis. The most commonly detected spirochetes were the pathogenic species B. intermedia and B. pilosicoli . The presumed nonpathogenic species B. innocens , B. murdochii , and the proposed “ B. pulli ” also were identified. Pathogenic B. alvinipulli was present in two flocks, and this is the first confirmed report of B. alvinipulli in chickens outside the United States. Brachyspira hyodysenteriae , the agent of swine dysentery, also was identified in samples from three flocks. This is the first confirmed report of natural infection of chickens with B. hyodysenteriae . Experimental infection studies are required to assess the pathogenic potential of these B. hyodysenteriae isolates.

The ban on chemotherapeutics against Histomonas meleagridis in the European Union has left turkey producers without an effective treatment against histomonosis. It has encouraged the development of alternative control methods amongst which are a number of herbal products. In the present study the in vitro effect of four herbal products against H. meleagridis was examined. After suspension of the herbal products in Dwyer's culture medium used for subculturing of a viable culture, parasite growth was monitored by frequently counting the histomonads until 72h of incubation. The solid products Aromabiotictrade mark, Enteroguardtrade mark, and Protophyt SPtrade mark as well as the positive control products dimetridazole and Histostat-50trade mark, were tested in twofold serial dilutions ranging in concentration from 400 to 12.5mug/mL, while the liquid product Protophyt Btrade mark was tested in concentrations ranging from 0.24% to 0.008% (v/v). The herbal products Enteroguardtrade mark and Protophyt Btrade mark as well as dimetridazole and Histostat-50trade mark, two chemotherapeutics with known antihistomonal activity, inhibited the growth of H. meleagridis, while no antihistomonal effect was found for Aromabiotictrade mark and Protophyt SPtrade mark.

C-profiling is a novel genotyping method for protozoan pathogens, based on polymerase chain reaction and sequencing of AT-rich Internal Transcribed Spacer-1 sequences. It was applied to various Histomonas meleagridis isolates originating from outbreaks of histomoniasis in six Dutch turkey and chicken flocks. Three different H. meleagridis genotypes were identified. Type I and type II were associated with clinical disease. In two flocks, both having recovered from an outbreak of histomoniasis, a type III strain was found that was also morphologically slightly different from the type I and type II isolates. C-profiling is a promising technique to differentiate between H. meleagridis subtypes, making it useful for epidemiological studies.

Dwyer's medium is a frequently employed culture medium for Histomonas meleagridis with rice powder as an essential ingredient. The effect of adding larger quantities of rice powder to the culture medium and the influence of the size of the rice particles on the growth of H. meleagridis was examined. Increasing the amount of rice powder from the standard amount of 10 to 12 mg to 50 to 100 mg per 12.5 ml medium resulted in approximately a 10-fold increase of parasites. Larger quantities of rice powder did not give better yields. The particle size of the rice powder proved relatively unimportant, although the addition of only large rice powder particles (>250 µm) resulted in a somewhat lesser yield. H. meleagridis cultures could be prolonged from approximately 4 days to at least 2 weeks without subculturing by supplementing the culture medium with rice powder only. Culture prolongee in vitro d'Histomonas meleagridis et recolte importante du parasite Le milieu de culture de Dwyer avec de la poudre de riz comm...

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is considered a hereditary cardiac disease, characterised by ventricular arrhythmias with left bundle branch block morphology, fibro-fatty replacement of cardiomyocytes and predominantly affecting the right ventricle [1, 2]. Recently, arrhythmogenic cardiomyopathy (AC) was suggested as the preferred terminology [3]. The most important considerations for this are that patients and families with ventricular arrhythmia and similar ARVD/C histopathological changes in the left ventricle have been recognised and described as left-dominant arrhythmogenic cardiomyopathy [4]. Mutations in desmosomal genes are associated with both ARVD/C and left-dominant arrhythmogenic cardiomyopathy and at the molecular level both ventricles and the interventricular septum are similarly affected [5, 6]. Classically AC is considered a disease of the desmosomes. Desmosomes are protein complexes located in the intercalated disk, important for mechanical integrity. For example, in approximately 60 % of Dutch AC patients a pathogenic desmosomal mutation is found, predominantly in the PKP2 gene [7]. Non-desmosomal genes have also been identified in families with phenotypical AC, for example TMEM43 [8]. Another mutation in a non-desmosomal gene is the p.Arg14del mutation in the phospholamban (PLN) gene. Phospholamban is involved in calcium homeostasis in the sacroplasmatic reticulum of the cardiac muscle cell [9]. Mutation carriers are phenotypically characterised by low-voltage electrocardiograms, ventricular arrhythmias, and contractile dysfunction involving both ventricles. Van der Zwaag et al. found that the PLN mutation was especially prevalent in the Netherlands, both in dilated cardiomyopathy (13 %) as well as in AC (12 %) [10]. This was a surprising finding because in other cohorts the prevalence of the PLN p.Arg14del mutation was found to be very low, between 0.08 % and 0.38 % [11]. In this issue of the Journal, Van der Zwaag et al. describe the origin of the PLN p.Arg14del founder mutation in the Netherlands [11]. This paper fits nicely in a series of articles started in 2009 on founder mutations in the Netherlands in different cardiomyopathies [12]. Founder mutations are mutations that emerged in a population many generations ago and have subsequently spread among following generations. In the study by Van der Zwaag et al. [11]. a large number of PLN p.Arg14del founder mutation carriers (probands and family members) were identified, in total 459 individuals. The authors analysed the origin of the PLN founder mutation first by haplotype analysis revealing that the mutation is estimated to be between 575 and 825 years old. Subsequently, in order to approximate the geographical origin of the PLN mutation the places of birth of the grandparents of the probands were plotted on a postal code map of the Netherlands, revealing that the majority of the grandparents came from the eastern part of the Dutch province of Friesland. The grandparents included in this study were born roughly between 1900 and 1925, i.e. approximately 75 to 100 years ago. At least six centuries of history are not accounted for, while in this time a lot happened. A quick search on the internet reminds us of the many wars the Dutch (including the ‘Friezen’) had with different enemies (e.g. Swedish, Spanish and Germans). The haplotype in the German family was the same as the haplotype found in the Dutch families, rising the possibility that the PLN founder mutation was introduced in Friesland by a German soldier, who possibly found a new love in Friesland. Also, due to migration, the residential area of the ‘Friezen’ around 1300 ran from the province of Noord-Holland up to Denmark. Therefore, it is still not sure that the origin of the mutation is in Friesland. A more detailed study of the migration in Friesland is needed to elucidate the exact place of origin of the PLN mutation; however, this is probably impossible, without the necessary registries. The authors subsequently studied the prevalence of the PLN p.Arg14del mutation in the PREVENT cohort, a large population-based cohort from the city of Groningen, in the north of the Netherlands (N = 8267). In this cohort six heterozygous PLN mutation carriers were found (0.07 %). As migration in the Netherlands has been very limited, as stated by the authors, the PREVENT cohort is possibly not the best cohort to test the true prevalence of the mutation in the Netherlands. Possibly the prevalence of the PLN founder mutation can be more accurately determined in other Dutch cohorts, not specifically including patients from the north of the country (e.g. ERGO, Rotterdam or SMART study, Utrecht). Of particular interest is the great difference in clinical expression in mutation carriers in the different families as shown by the family presented by the authors. This phenomenon is present in many different cardiomyopathies (e.g. hypertrophic cardiomyopathy) and primary electric cardiac diseases (e.g. long-QT syndrome). Different possible answers are the presence of polymorphisms influencing the calcium handling in the cardiac myocyte or related genes. Recently, the down-regulation of the cardiac sodium channel was found to be important in arrhythmogenesis in plakophylin-2 haploinsufficient mice13 and is possibly a predictor for SCD in patients with AC, making SCN5A another possible modulating gene, for example. Furthermore, differences between family members in the susceptibility for fibrosis in the heart could be of importance. Finally, there might be regions in the Netherlands with specific ‘environmental influences’ that make people more susceptible for developing a more severe phenotype. As mentioned before, in the PREVENT cohort six carriers of the PLN mutation were found and one of them developed heart failure within 10 years of follow-up (17 %). The question is whether it is mandatory to identify all carriers of the PLN mutation to be able to screen these individuals for risk factors for sudden cardiac death and/or heart failure. As the authors already state the PHORECAST study is an important tool to investigate the questions about risk stratification for sudden cardiac death and development of heart failure, consequently these findings should give rise to treatment strategies to prevent these severe complications. In conclusion, the PLN p.Arg14del founder mutation appears to originate from the north of Friesland. In the near future the goal should be to identify all carriers of the PLN p.Arg14del mutation to be able to prevent SCD and possibly treat asymptomatic carriers to prevent progression to disease.

Histomona meleagridis is a protozoan parasite that may cause outbreaks of histomonosis with high mortality, especially in turkey flocks. Chickens are less susceptible to the disease than are turkeys, but are considered to act as an important reservoir. To determine the seroprevalence of H. meleagridis in Dutch layer chicken flocks, a large scale seroepidemiologic study (3376 samples) was performed by sampling 12 organic flocks, 24 free-ranging flocks, 40 flocks with floor housing, and 40 flocks with cage housing. At the end of the laying period, approximately 30 blood samples per flock were collected for serology. The seroprevalence found was high. In every flock, at least one of the samples tested positive while in 87% of the flocks, at least one of the samples was strongly positive. There were no significant statistical differences in seropositivity between the housing types. To confirm the enzyme-linked immunosorbent assay (ELISA) results, a small-scale seroepidemiologic study (576 samples) was performed in 29 additional layer chicken flocks kept in different housing systems. Subsequently, a subset of five seropositive flocks was selected. Five birds were obtained from each of these flocks in order to detect the parasite using culture and PCR. In all five flocks, H. meleagridis was either isolated from (culture), detected in (PCR), or both, the birds sampled. Together with the previously performed validation studies, the latter results confirm that the positive ELISA serology found is genuine. We conclude that the seroprevalence of H. meleagridis in layers is, as anticipated, high.

Patients who have had an episode of symptomatic venous thromboembolism are usually treated for at least five days with intravenous unfractionated heparin or subcutaneous low-molecular-weight heparin. Thereafter, they received a three month course of a vitamin K antagonist, with a dose adjusted to achieve an International Normalized Ratio between 2.0 and 3.0. Some patients have contraindications to vitamin K antagonists. In addition, treatment with vitamin K antagonists has the disadvantage of regular laboratory measurements. The objective of this review was to evaluate the efficacy and safety of long-term treatment of symptomatic venous thromboembolism with low-molecular-weight heparins compared with vitamin K antagonists. Computerized searches of MEDLINE, EMBASE and Current Contents were made and relevant journals were hand-searched using the search strategy described by the Cochrane Peripheral Vascular Disease Group. In addition, randomized clinical trials were located through personal communication with colleagues. Where necessary, the reviewers contacted pharmaceutical companies for further information. Two reviewers evaluated studies independently for methodological quality. Two reviewers reviewed and extracted data independently using a standard form. Primary analysis concerned all patients in the studies during the period of randomized treatment. Additional separate analyses were performed for category I and category II studies; studies that used similar initial treatments in both study arms and those that used different treatment regimes during the initial treatment; and the total period of follow-up in the different studies. Five studies were identified that fulfilled our predefined criteria (three category I and two category II studies). When all five studies were combined, a statistically non-significant reduction of the risk of recurrent symptomatic venous thromboembolism in favor of low-molecular-weight heparin treatment (OR 0.72; 95% CI [0.42, 1.23]) was found. In category I studies, analysis of the pooled data showed a statistically non-significant reduction of the risk of recurrent symptomatic venous thromboembolism in favor of low-molecular-weight heparin treatment (OR 0.75; 95% CI [0.40, 1.39]). This OR was mainly due to one possibly confounded study, and after omitting this study from the analysis a statistically non-significant reduction of the risk of recurrent symptomatic venous thromboembolism in favor of vitamin K antagonist treatment remained (OR 1.95; 95% CI [0.74, 5.19]). No differences in the risk of bleeding (OR 0.63; 95% CI [0.21, 1.88]) and mortality (OR 1.13; 95% CI [0.47, 2.69] were observed. Low-molecular-weight heparins are possibly as effective and safe as vitamin K antagonists in the prevention of recurrent symptomatic venous thromboembolism after an episode of symptomatic deep venous thrombosis, but have the disadvantage of much higher medicinal costs. Treatment with low-molecular-weight heparin is possibly a safe alternative in some patients; for example patients who live in geographically inaccessible places; patients who are reluctant to go to the thrombosis service on a regular basis; and patients with contraindications to vitamin K antagonists (e.g. pregnant women). Therefore, in the absence of definitive evidence on the safety and efficacy of low-molecular-weight heparins compared with vitamin K antagonists, we believe that treatment with vitamin K antagonists is still the treatment of choice in the prevention of recurrent symptomatic venous thromboembolism after an episode of deep venous thrombosis, in the majority of patients

An international ring trial for Salmonella-ELISAs for swine serology was organized. Twelve laboratories participated and used "in-house" ELISAs or commercially available kits. In total 47 well-defined sera from various sources, including inoculation studies with Salmonella-strains from sero-groups B, C1, C2, D, and E1, were tested blindfold. The specificity of most ELISAs was satisfactory, but relatively large differences were found between the sensitivities of the tests. It is concluded that international reference samples should be made available to guarantee a minimum level of sensitivity.

Low-molecular-weight heparin compounds have been used in the treatment of patients with venous thromboembolism for approximately 15 years. Ever since their introduction, there has been discussion about whether low-molecular-weight heparin compounds differ in their efficacy and safety. The best answer would be provided by direct comparison of different low-molecular-weight heparin preparations; however, these trials have not been conducted. Classical meta-analysis has its limitations for such a comparison since only a very small number of trials with the respective low-molecular-weight heparin compounds are available. The objective of the present analysis has been the use of meta-regression to compare the efficacy and safety of different low-molecular-weight heparin compounds in the initial treatment of patients with venous thromboembolism. We used computerized literature searches to identify studies that compared dose-adjusted unfractionated heparin treatment with fixed dose subcutaneous low-molecular-weight heparin treatment in patients with established venous thromboembolism. The individual odds ratios of the studies were plotted against the absolute percentage of the major outcomes in the unfractionated heparin control group. Linear regression was used to find differences between different low-molecular-weight heparin compounds. There appears to be some variation in efficacy and safety among the currently available low-molecular-weight heparin preparations.

Reports on the effects of mange on the production performance of pigs are conflicting. So far, studies have used experimental infections, by depositing encrusted lesions from chronically infected pigs into the ears of experimental pigs. However, this is a poor representation of what happens under natural field conditions. The purpose of our study was to quantify the effects of sarcoptic mange on production performance and pruritus in pigs that were infected by contact with S. scabiei var. suis-infected pigs. A total of 80 piglets were matched by sex and weight and randomly divided between experimental and control compartments. In the experimental compartment, each of three naturally S. scabiei var. suis-infested pigs were randomly allocated to three pens with 13 susceptible pigs each. From day 0 to 35, the growth performance of pigs in the experimental compartment was significantly (P=0.04) worse (35 g/d) than of pigs in the control compartment. From day 35 to 112, there was a statistical trend (P=0.10) that the growth performance of pigs in the experimental compartment was lower (50 g/d) than that of pigs in the control compartment. For the complete fattening period (0-112 or more days), the growth performance of pigs in the experimental compartment was significantly (P=0.05) worse (41 g/d) than that of pigs in the control compartment. Mean feed conversion ratio (kg feed per kg gain) was 2% higher in the experimental compartment compared with the control compartment. Pigs in the experimental compartment had a nine times (95% CI: 2 - 44) higher chance of showing pruritic behaviour than pigs in the control compartment.

Background Pathogenicity of truncating or splice site mutations in arrhythmogenic right ventricular cardiomyopathy (ARVC) patients has been widely accepted. However, pathogenicity of missense variants remains controversial. We assessed pathogenicity of ARVC-related missense variants and associated phenotype. Methods Pathogenic mutations were found in 571 patients (239 families, age 43 ± 18 years) from US/Dutch ARVC cohorts by screening of desmosomal (PKP2, DSC2, DSG2, DSP, JUP) and/or nondesmosomal (PLN, TMEM43) genes. Patients with >1 mutation or variant were excluded. Missense variants were considered pathogenic when (1) algorithms SIFT and PolyPhen-2 predicted pathogenicity (SIFT: tolerance index score ≤0.05, PolyPhen-2: pathogenicity score >0.90) and (2) minor allele frequency in 1000 genomes was Results A pathogenic missense mutation was found in 86/571 patients (15%). Frequently occurring mutation was PKP2 c.2386T>C (p.Cys796Arg; 34/86). Survival curves (P = .065) and, more importantly, sustained arrhythmia (ventricular tachycardia/fibrillation or appropriate ICD therapy) free survival curves (P = .810) of patients with a missense mutation were similar to those with pathogenic non-missense (330/571 truncating, 129/571 splice site) mutations (Figure). Conclusions ARVC-related missense mutations have a similar pathogenic character as non-missense mutations regarding survival and substantial arrhythmic burden. Pathogenicity of missense mutations can be correctly predicted by combined criteria from SIFT, PolyPhen-2, and 1000 genomes. Download : Download high-res image (120KB) Download : Download full-size image

PURPOSE: With the increased use of genetic testing for arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C), this disease is being increasingly recognised among elderly patients. However, elderly ARVD/C patients were underrepresented in prior cohorts. We aimed to describe the phenotypical characteristics and outcomes among ARVD/C patients surviving ≥50 years. METHODS: We assessed detailed phenotypical data of 29 patients who (1) presented at ≥50 years of age; and (2) fulfilled 2010 Task Force Criteria (TFC) for ARVD/C by last follow-up. Primary outcome was the occurrence of a major ventricular arrhythmia (sudden cardiac death, resuscitated sudden cardiac arrest or sustained ventricular tachycardia). RESULTS: The majority (55 %) of elderly ARVD/C subjects were male, with a mean age of 59.0 ± 5.8 years at presentation. Study participants fulfilled a median of six (IQR 5-8) TFC criteria by last follow-up, of which arrhythmia criteria were most frequent (97 %), followed by structural criteria (83 %), depolarisation criteria (72 %) and repolarisation criteria (69 %). By last follow-up, 15 (52 %) patients had experienced major ventricular arrhythmias. Most patients (n = 12) presented with this arrhythmia, while three experienced the event during 5.4 ± 3.2 years of follow-up. Compared with patients without an arrhythmic event, patients with major arrhythmias were more likely to be proband (p < 0.001) and male (p = 0.042). Likewise, survival free from sustained ventricular arrhythmia was lower among probands and males. CONCLUSION: Phenotypic characteristics of elderly ARVD/C patients are characterised by depolarisation abnormalities and structural cardiac changes. Ventricular arrhythmias in this elderly cohort are associated with male gender and proband status.