The importance of the adrenal gland in regard to lactation and reproduction in cattle has been recognized early. Caused by interest in animal welfare and the impact of stress on economically important traits in farm animals the adrenal gland and its function within the stress response is of increasing interest. However, the molecular mechanisms and pathways involved in stress-related effects on economically important traits in farm animals are not fully understood. Gene expression is an important mechanism underlying complex traits, and genetic variants affecting the transcript abundance are thought to influence the manifestation of an expressed phenotype. We therefore investigated the genetic background of adrenocortical gene expression by applying an adaptive linear rank test to identify genome-wide expression quantitative trait loci (eQTL) for adrenal cortex transcripts in cattle.CYP27A1 and LHCGR that have been shown to affect economically important traits in cattle.A total of 10,986 adrenal cortex transcripts and 37,204 single nucleotide polymorphisms (SNPs) were analysed in 145 F2 cows of a Charolais × German Holstein cross. We identified 505 SNPs that were associated with the abundance of 129 transcripts, comprising 482 cis effects and 17 trans effects. These SNPs were located on all chromosomes but X, 16, 24 and 28. Associated genes are mainly involved in molecular and cellular functions comprising free radical scavenging, cellular compromise, cell morphology and lipid metabolism, including genes such as In this study we showed that adrenocortical eQTL affect the expression of genes known to contribute to the phenotypic manifestation in cattle. Furthermore, some of the identified genes and related molecular pathways were previously shown to contribute to the phenotypic variation of behaviour, temperament and growth at the onset of puberty in the same population investigated here. We conclude that eQTL analysis appears to be a useful approach providing insight into the molecular and genetic background of complex traits in cattle and will help to understand molecular networks involved.The online version of this article (doi:10.1186/s12863-016-0442-x) contains supplementary material, which is available to authorized users. Stress-related effects on productivity –9, healtAdrenal cortex is a promising target to investigate these effects, due to its impact on stress response and regulation of essential metabolic pathways . Human dTherefore we investigated the genetic background of adrenocortical gene expression in a F2 cattle population (SEGFAM). The SEGFAM population is a cross of Holstein cows that were selected for high milk yield and Charolais bulls that were selected for meat production for a long time. By crossing the two breeds a high genetic and phenotypic variance in regard to milk and meat production traits was expected due to segregation of different QTL alleles in the F2 cross . The popwww.qiagen.com/ingenuity) and literature reviews to identify the molecular pathways involved and evaluate potential targets for further research.The aims of this study were first to identify eQTL in the adrenal cortex in cattle and secondly, to evaluate whether associated genes and molecular pathways are known to affect economically important traits in cattle. The analysed data comprised genotypes of 37,204 SNPs and expression values for 10,986 transcripts. In order to account for systemic effects and to de-correlate the expression data a sire-dam model was used during pre-processing. To account for relatedness the complete pedigree data comprising the founder and F1 generation was used. Then we performed a genome-wide eQTL analysis using an adaptive linear rank test  on the rp-values of the adaptive linear rank test we also included the unadjusted p-values of adaptive linear rank test, Kruskal Wallis-test and ANOVA for completeness. The number of eQTL per chromosome is shown in Fig. In total, 505 SNPs were identified to be associated with the expression of 129 transcripts. A complete list of eQTL including the chromosomal position and annotation of the SNP and the gene encoding the transcript is provided in Additional file LYPD6B (Chr. 2), GRIP1 (Chr. 5), SNCB (Chr. 7), COQ3 (Chr. 9) and ALDH5A1 (Chr.23), for example, was associated with 21, 15, 35, 28 and 20 SNPs, respectively. These SNPs were located up to 30 MB up- or downstream of the gene encoding the transcript. Considering that we investigated a F2 population that showed considerable linkage disequilibrium  compared with trans eQTL (17) observed in our study is high. This is in line with the observation that the number of identified local or cis eQTL is related to sample size, extent of linkage disequilibrium, size of the window applied for cis eQTL and effect size of eQTL which is assumed to be stronger for cis eQTL in comparison with trans eQTL . A reasoIngenuity® pathway analyses of genes encoding affected transcripts revealed that some of the genes are involved in “Diseases and Bio Functions” like lipid metabolism, inflammatory response and reproductive system development and function. This indicates that the identified eQTL potentially contribute to the phenotypic variation of economically important traits in cattle. For some of the genes there is evidence that they have an effect on reproduction, milk performance, growth or health traits, based on genetic and molecular studies in cattle and other farm animals. The following sections comprise details for selected genes. In addition, comparisons with studies within the SEGFAM population indicate that some of the genes affected by eQTL contribute to the phenotypic variation of behaviour, temperament and growth at the onset of puberty. Since the effects of adrenocortical gene expression on most of the economically important traits in cattle still have to be investigated, for other genes only the contribution to disease phenotypes in human or mice or the involvement in specific metabolic pathways like reproductive system development and function suggests that they might contribute to the phenotypic variation of economically important traits in cattle. In addition, the sheer existence of genomic regions affecting the adrenocortical transcript abundance of genes that are known to influence economically important traits in cattle is no evidence that the adrenocortical transcript abundance is causal, but similar effects might be present in other organs and tissues  providinVimentin (VIM) is a type III intermediate filament that is expressed in cells of mesenchymal origin and has key functions in the formation of the cytoskeletal network, organelle positioning, cell migration and adhesions as well as in cell signaling [VIM expression has been shown to be required for the development and establishment of the embryo in cattle. It was reported to be differentially expressed between nuclear transfer and by in vitro fertilization produced embryos [ignaling . VIM exp embryos , 34. Now embryos , 36.luteinizing hormone/chorigonadotropin receptor (LHCGR) is a G-protein-coupled receptor that is mainly known for binding luteinizing hormone and chorionic gonadotropin and mediating their cellular actions. In humans inactivating mutations in the LHCGR gene have been shown to cause infertility in both male and female [LHCGR is discussed to be involved in the manifestation of the pregnancy induced Cushing’s Syndrome due to adrenocortical LHCGR overexpression that may lead to adrenocortical hyperfunction [LHCGR gene have been shown to be associated with calving interval, days to first service and milk yield [The d female . In addifunction –40. In clk yield  as well lk yield –44 that lk yield , 36.cytochrome P450, family 27, subfamily A, polypeptide 1 (Cyp27A1) is a key-enzyme in Vitamin D3 metabolism [LHCGR, Cyp27A1 has also been discussed to be important for reproduction [The tabolism , 47. In tabolism . In a prtabolism . In addioduction  but has xanthine dehydrogenase (XDH) is a xanthine oxidoreductase that belongs to the group of molybdenum iron-sulfur flavin hydroxylases. XDH contributes to the detoxification of endogenous or xenobiotic compounds and is involved in the oxidative metabolism of purines [XDH has been shown to be upregulated during the lactation cycle due to its contribution to lipid droplet formation [XDH protein abundance has been shown to increase in the first nine days of lactation in milk serum [XDH to be a candidate gene for milk production and mastitis susceptibility based on studies in mouse and cattle.The  purines , 52. In ormation . In addilk serum , and [55Transferrin (TF) together with ferritin is important for acquisition, transport and storage of iron which is essential for many metabolic processes like the synthesis of hemoglobin for oxygen transport [TF has been shown to be highly polymorphic [TF polymorphisms are associated with protein yield, 305-day milk yield and mastitis susceptibility. In addition, they showed that TF mRNA expression was higher in mastitis affected in comparison with unaffected mammary tissue [ransport . In cattymorphic  and a sty tissue .aldehyde dehydrogenase 5 family, member A1 (ALDH5A1) is a semialdehyde dehydrogenase that is important for the metabolism of γ-aminobutyric acid (GABA) the major inhibitory neurotransmitter in the brain [ALDH5A1 has been reported in Angus cattle selected for high or low residual feed intake [ALDH5A1 is located on chromosome 23 in the genomic region were bovine MHC genes are located. In this genomic region we identified a high number of SNPs that were not only associated with the expression of ALDH5A1 but also with the expression of some MHC genes like the MHC class I heavy chain BOLA and the major histocompatibility complex BOLA-DQA1. This genomic region has been reported to be associated with the antibody mediated immune response in Canadian Holstein cows [The he brain . A diffed intake . Increasd intake  and an ad intake . 2,4-dihd intake  and a ted intake . Interesein cows .NME6 and RAB32 in this study. The same SNPs were previously identified to be associated with the inactivity in an open field test and the explorative behaviour in a novel object test, respectively [Another study analyzed the impact of genetic variants on behaviour characteristics assessed in an open field and novel object test within the SEGFAM population . We idenectively .NME/NM23 nucleoside diphosphate kinase 6 (NME6) is a nucleoside diphosphate kinase. Little is known about its function besides that it is assumed to play a role in cell growth and cell cycle progression [NME6 is located in a QTL region associated with back fat [The gression , 66. In back fat .RAB32, member RAS oncogene family (RAB32) belongs to the RAB family of small GTP-binding proteins. It is involved in the biogenesis of lysosome-related organelles like melanosomes [NME6 we could not find any additional information other than a suggested role in lipid metabolism [anosomes . Similartabolism .PRL, ESRRG and GNRH1 as upstream or master regulators, providing insight into regulatory mechanisms that are potentially affected by eQTL.The upstream analysis revealed Prolactin (PRL) is an anterior pituitary protein hormone that is not only important for reproduction but is also involved in the control of behaviours [PRL in regard to lactation and milk production have been investigated. In the last years several studies in cattle investigated the inhibition of PRL-release on milk yield and showed that the inhibition was associated with a decrease in milk yield additionally indicating an important role of PRL in the maintenance of lactation [haviours . In cattactation –73. The actation .Gonadotropin-releasing hormone 1 (GNRH1) encodes a protein that is secreted and cleaved to form the luteinizing hormone-releasing hormone and the prolactin release-inhibiting factor that regulate LH and FSH gene expression [LH and FSH both have been early recognized to be involved in the onset of puberty in cattle and today Gonadotropin-releasing hormone is used to synchronize cows for timed artificial insemination especially in beef cattle [pression . LH and f cattle , 76. In f cattle .Estrogen-Related Receptor Gamma (ESRRG) that belongs to the estrogen receptor-related receptor family has recently been shown to be important in the fibrate-mediated regulation of lipid metabolism genes in a human ApoA-I transgenic mice model [ESRRG was also identified to be associated with the onset of puberty in cattle [The ce model . Interesn cattle .In the present study we investigated the genetic background of adrenocortical gene expression and identified eQTL affecting the expression levels of genes that have been reported to influence economically important traits in cattle. In addition, some of the genes and molecular pathways affected by eQTL were previously shown to contribute to the phenotypic variation of behaviour, temperament and growth at the onset of puberty in the SEGFAM population. Therefore, eQTL analysis appears to be a useful approach to gain insight into the molecular and genetic background of complex traits in cattle. Additional studies are needed to identify the causal polymorphisms and underlying molecular mechanisms (cis or trans) and the effects of genetic variation on gene expression in other tissues.This study comprised 145 F2 cows from the experimental resource cross population (SEGFAM) that was initiated at the Leibniz Institute for Farm Animal Biology (FBN) in the P0 generation by crossing five purebred Charolais sires to purebred German Holstein cows . The popSNP genotyping was accomplished using Illumina® BovineSNP50 Beadchip v1.0 and v2.0. Genotype calling was performed with the Genotyping Module of the GenomeStudio V2011.1 Software (Illumina®). For this purpose genomic DNA was extracted from mammary gland tissue with the QIAmp DNA Mini Kit  and prepared for hybridization on the Illumina® BovineSNP50 Beadchip following manufacturer’s instructions. Genotype calling and quality control was performed according to  for v1.0Total RNA was isolated from the adrenal cortex of 145 animals using TRI Reagent . After DNase I treatment the RNA was further purified with the RNeasy Kit  following the manufacturers recommendations. RNA was quantified using the NanoDrop ND-1000 spectrophotometer . Integrity of RNA was checked by running 1 μg of RNA on a 1 % agarose gel. Absence of DNA contamination was verified by PCR amplifying fragments of GAPDH with RNA as a template. For hybridization, 500 ng of total RNA were amplified using Ambion WT Expression Kit (Affymetrix). Subsequently, 5.5 μg of the resulting cDNA was fragmented and labeled using the Affymetrix Terminal Labeling Kit. The fragmented cDNA was hybridized to the microarray using the Affymetrix Hyb-WashStain Kit and Affymetrix standard protocols. Fluidic station protocol was FS450_0001.For expression profiling the custom GeneChip Bovine Gene v1 Array was used. Affymetrix designed the GeneChip Bovine Gene v1 Array based on Ensemble and RefSeq predictions for the Genome Bos Taurus Build 4.0. The design was targeted to develop a whole Genome Expression Array with approximately 25 probes per transcript distributed over the whole length of each transcript. In total 194,712 probe sets targeting at approximately 24,000 transcripts are implemented on the array. In addition standard Affymetrix controls for hybridization and labeling efficiency as well as for non-specific binding are included on the array. Gene-level analysis was performed using Affymetrix® Expression Console™ Software. Microarray raw data were preprocessed using the RMA algorithm . QualityAfter quality control and filtering the expression data was further pre-processed to account for systemic effects. Furthermore, the statistical test used for eQTL analysis required the data to be decorrelated. Since we expected gametic phase disequilibrium for some of the loci, we refrained from estimating kinship from SNP data but preferred the use of kinship estimates from pedigree information. Therefore the following sire-dam model was applied in ASReml  to accouyi is the expression value for transcript y of animal i , sj is the fixed effect of year and season  at slaughter, xi is the age in days of animal i at slaughter, aif is the additive-genetic effect of the sire of animal i, aim is the additive-genetic effect of the dam of animal i and εi is the random residual effect. To estimate the additive-genetic effects the complete pedigree data comprising 38 P0 and 52 F1 animals was used. The residuals comprise the Mendelian sampling effect as an uncorrelated genetic component as well as a random deviation. For further analysis the residuals were considered as de-correlated expression data.where p-values [−7 for unadjusted p-values. This global false discovery rate (FDR) threshold of 0.1 is less conservative for trans associations than usual, but it is very conservative for cis associations, since it corresponds to a FDR threshold of approximately 0.0035 for an eQTL analysis in which only cis associations (approximately 13 million) are considered for the calculation of the Benjamini-Hochberg adjusted p-values. This was done because the majority of associations was expected between SNPs and transcripts for which the encoding gene of the transcript was located on the same chromosome in close vicinity to the SNP.For eQTL analysis an adaptive linear rank test  was applp-values  (considewww.qiagen.com/ingenuity). The purpose of the functional analysis was to determine if the 129 identified genes are statistically significant over-represented (more genes than expected by chance) for a specific molecular or cellular function in comparison to a reference-set of genes. Similar to the functional analysis, the Ingenuity® upstream analysis was performed to measure whether there is a statistically significant overlap between the 129 identified genes and the genes that are regulated by a specific transcription regulator. For both tests IPA uses a Fisher’s Exact Test to determine the significance of the over-representation or overlap in comparison to a reference set. As reference set for the functional analysis the Ingenuity® Knowledge Base was chosen, because the reference network that is used to identify the upstream and master (upstream and downstream) regulators in the upstream analysis is based on the Ingenuity® Knowledge Base. Reported p-values are Benjamini-Hochberg adjusted.The functional and upstream analyses were performed through the use of QIAGEN’s Ingenuity® Pathway Analysis (IPA, QIAGEN Redwood City,


Minimal hepatic encephalopathy (MHE) is clinically undetectable and the diagnosis requires psychometric tests. However, a lack of clarity exists as to whether the tests are in fact able to detect changes in cognition.To examine if the continuous reaction time test (CRT) can detect changes in cognition with anti-HE intervention in patients with cirrhosis and without clinically manifest hepatic encephalopathy (HE).Firstly, we conducted a reproducibility analysis and secondly measured change in CRT induced by anti-HE treatment in a randomized controlled pilot study: We stratified 44 patients with liver cirrhosis and without clinically manifest HE according to a normal (n = 22) or abnormal (n = 22) CRT. Each stratum was then block randomized to receive multimodal anti-HE intervention (lactulose+branched-chain amino acids+rifaximin) or triple placebos for 3 months in a double-blinded fashion. The CRT is a simple PC-based test and the test result, the CRT index , describes the patient’s stability of alertness during the 10–minute test. Our study outcome was the change in CRT index in each group at study exit. The portosystemic encephalopathy (PSE) test, a paper-and-pencil test battery , was used as a comparator test according to international guidelines.The patients with an abnormal CRT index who were randomized to receive the active intervention normalized or improved their CRT index . Additionally, their PSE improved . There was no such effect in any of the other study groups.In this cohort of patients with liver cirrhosis and no manifest HE, the CRT identified a group in whom cognition improved with intensive anti-HE intervention. This finding infers that the CRT can detect a response to treatment and might help in selecting patients for treatment. The term minimal hepatic encephalopathy (MHE) denotes cognitive deficits in a patient with liver disease that are undetectable at clinical encounters. A MHE diagnosis, therefore, requires psychometric tests. The prevalence of MHE is very high in cirrhosis patients; i.e., present in approximately 50%, 2. The Despite the positive effects of diagnosing and treating MHE, only a few centres do so routinely. One impThe underlying problem is that there is no gold standard for the diagnosis or grading of MHE. In specialist clinical centres, a few psychometric tests are used routinely in different combinations to diagnose MHE, but the test results are widely discordant in the individual patients –7—althouWe took advantage of the clinical reports on positive effects of MHE treatments to utilize anti-HE intervention as a tool for validation of a psychometric test: the continuous reaction time (CRT) test , 8, 9. TThe psychometric test we evaluated in this way, the CRT, is a simple PC-based performance test measuring the important cognitive ability of being able to deliver stable motor reactions to sensory stimuli—an ability of obvious importance for every-day living. In Scandinavia, this test is established for clinical routine use in the management of MHE. In the pilot study we present here, we also applied the portosystemic encephalopathy (PSE) test, a paper-and-pencil test recommended as an international comparator test for MHE studies.Our hypothesis was that the CRT is able to identify patients with liver cirrhosis who improve cognition by anti-HE intervention. The aim of the study was to measure the CRT before and after such intervention vs. placebo.Twenty-two patients with liver cirrhosis underwent repeated CRT testing to assess CRT reproducibility. They were included from 2 Danish sites  during October 2012 without clinically manifest HE and were all tested twice before and twice after noon on two consecutive days reaching a total of 8 measurements per patient within 48 hours. Patients were aged 61 years (range 42–73 years), 18 were men, mean MELD score was 6.12 (range 6–16) and all had alcoholic cirrhosis. All patients were tested between 8.00 and 14.00 in an undisturbed location by one of 3 trained operators. All participants gave their informed written consent and The Regional Scientific Ethical Committee for Southern Denmark approved the study protocol (Protocol number S-20120196). In- and exclusion criteria cf. below.The inclusion criteria were age above 18 years, diagnosis of liver cirrhosis and absence of clinically manifest HE. From November 2013 to December 2014, we enrolled 44 such patients from the Hospital of Southwest Jutland (n = 23) and Odense University Hospital n = 21; . They wewww.Bitmatic.com, Aarhus, Denmark). After a 2–minute instruction, the patient is left alone and undisturbed to complete the 10-minute test during which the patient responds to 150 sound stimuli (beeps at 500 Hz and 80 dB) by pressing a button. They are delivered at random intervals from 2 to 6 seconds and the patient is instructed to press the button as soon the beep is heard. The software registers the response times and calculates the CRT index (the ratio: 50 percentile/[90 minus 10 percentile]). The method measures and combines motor reaction speed, sustained attention, and inhibitory control, which are all key abilities in daily life functioning [The test is PC-based and requires a standard laptop, simple software, headphones, and a handheld trigger button , Number Connection Test A (NCT-A), Number Connection Test B (NCT-B), Serial Dotting Test (SDOT), and Line Tracing Test . Completing all sub-tests takes approximately 15 minutes and the presence of staff for guidance and timing is required. Calculation of the final test result using age-adjusted norms takes another 5 minutes. The test score is calculated from the time (seconds) spent on each sub-test, which is converted into a score that ranges between -3 and 1. The LTT is converted into 2 scores—a time score and an error score. A score of 0 is given for a performance within +/- 1 SD. The total test score, the portosystemic hepatic encephalopathy score (PHES), is the sum of the scores. A result below -4 is abnormal and may indicate MHE in liver patients. Danish norm data  were applied. The test measures attention, working speed and accuracy, visual orientation, and visuo-spatial construction .After initial testing and stratification according to the CRT index, the patients were block randomized into two groups to receive either triple-active anti-HE intervention or triple placebo for 3 months. The patients randomized to active intervention received 66.7% lactulose syrup , branched-chain amino acids  and rifaximin . The triple placebo regimen consisted of placebo to lactulose (50% glucose syrup), placebo to branched-chain amino acids  and placebo to rifaximin . Patients received oral and written information about possible adverse effects of the medications. The effect of the interventions was measured after study end. A protocol summary is available in The Odense University Hospital Pharmacy boxed active and placebo medications in numbered packages and intervention assignment was obtained from a computer-generated randomization list in blocks of 22. All items were labelled with only the patient and study number (numbers 1–44). Boxes numbered 1–22 contained 11 sets of active medicine and 11 sets of placebo medicine in random sequence and were consecutively assigned to the stratum of participants with a CRT index < 1.9. The boxes numbered 23–44 likewise contained the same sets as the first boxes and were sequentially assigned to the stratum of participants with a CRT index greater than or equal to 1.9. In this way, all the patients, including those with a CRT index within the normal range (up to a CRT index of 2.5), randomly received active intervention or placebos. The study participants and all staff were blinded until all data were collected.There were no reported serious adverse effects for any of the components of the active and placebo interventions. We considered it ethically acceptable to administer the interventions to both cirrhosis patients with and without psychometric signs of MHE and without the aim of documenting a clinical outcome benefit. The potential benefit to future patients lies in the prospective of improving the possibilities of identifying such patients who can be expected to improve their cognition by treatment. The study principle, methods and protocol complied with the 1975 Declaration of Helsinki and was approved by the Danish National Committee on Health Research Ethics and registered at clinicaltrial.gov (Identifier: NCT01773538). All participating patients gave their informed written consent.RCT data and reproducibility data are available in change between active and placebo groups. We wanted a type I error of 5% and a type II error of 15% (power 85%). The expected dropout rate was set at 20%. We assumed that the standard deviation of repeated measurements is a reasonable estimate of the unknown standard deviation of changes in CRT index and the desired effect size was set at 0.68. We found that 6 patients were needed per group. This number was increased to 9 as the true SD of changes in CRT index most likely is larger. As we ultimately decided to have 2 stratae in each group we included 36 patients plus 8 to take drop outs in to account i.e. a total of 44–22 in each treatment arm [For the RCT our hypothesis was that patients randomized to active intervention would obtain an improvement in the CRT index significantly greater than that resulting from the placebo intervention. Accordingly, our primary outcome was the difference in mean CRT index ment arm . The CRTment arm . StudentResults are displayed in Seventeen patients with an abnormal CRT index below 1.9  and 16 with a normal CRT index ≥1.9  completed the 3-month study .Baseline characteristics are shown in The stratum with an abnormal baseline CRT index: Baseline characteristics are displayed in The stratum with a normal baseline CRT index: No change from the baseline CRT index was found in the active or the placebo group. The PSE test also did not change. There was no difference in CRT and PSE changes .Ammonia and manifest HE episodes: The P-ammonia concentration decreased on average by 22%  in the group with an abnormal entry CRT index and randomized to active intervention, but such an effect was not observed in any other group. No significant or systematic change in Child Pugh, MELD, or SIP score was observed in either strata or group. CRP remained unchanged in all groups.During the post–study follow-up, 2 out of 13 in the group receiving active intervention and 4 out of 20 in the placebo group experienced an episode of manifest HE. In the active group, 1 patient died (acute-on-chronic liver failure) while 5 patients in the placebo group died .Post hoc analysis of psychometric effects according to entry PSE: The patients were primarily stratified according to their CRT index, so the analysis according to PSE is an exploratory secondary analysis. It is based on 25 patients with an abnormal entry PSE, 13 of them receiving the active intervention and 12 of them receiving the placebos. Those on the active intervention improved their CRT index from 1.6±0.4 to 2.2±0.5 (p = 0.02) and PHES from -8.9±3.2 to -5.0±4.4 (p = 0.0014). No improvement in PHES or CRT index was observed in the placebo group .The overall dropout rate was 25% (n = 11) and thus 5% larger than expected. Nine (40%) dropped out in the group randomized to active invention and two (9%) in the placebo group (p = 0.08). Patient characteristics and reasons for dropout are listed in The purpose of this pilot study was to evaluate if the CRT test is able to detect and predict an effect of anti-HE-treatment. The central finding was that the cirrhosis patients without manifest HE and with an abnormal CRT index normalized or improved their psychometric status i.e. CRT index (and PHES) with three months of triple-active anti-HE intervention—this did not occur in any other study group. The clinical implication of this finding it is that the CRT test might be useful in detecting an effect of anti-HE treatment (and useful in identifying cirrhosis patients who will benefit cognitively from anti-HE treatment). The test thus could be used both in the daily clinic and as an outcome measure in future scientific studies.not primarily aim to test the intervention effect. To secure the presence or non-presence of the desired psychometric signal, we applied the expectedly most powerful anti-HE intervention viz. the multi-modal triple modality. It is possible that we would have reached the same result with lactulose as a mono-intervention as there is now evidence that lactulose does ameliorate MHE, albeit the diagnostic methods vary among studies [The methodological novelty of our study is that we used the effects of reportedly efficient therapeutic interventions in a randomized controlled fashion as a means to validate the meaningfulness of the CRT method in the management of MHE. Our primary outcome was the changes in psychometry i.e. the CRT index whereas we did  studies . The cur studies . The resAdditionally, the comparator measure, the PSE, improved in the group randomized to active intervention as whole  and the No other psychometric validation study within this research field is based on the same approach. It is noteworthy that the patients with an abnormal CRT index who received the placebo intervention tended to improve their CRT index—only less so than the patients on the active intervention (by average 0.25 vs. 0.92). This illustrates the time-dependence of the cognitive disturbances cause by MHE and emphasizes the necessity for randomized intervention for evaluation of the test. As could be expected, there were no such temporal dynamics in the patients with a normal entry CRT index.a priori assumptions after which the study was dimensioned. Its robustness is supported by the fact that the outcome effect was more than threefold larger than the intra-individual day-to-day CRT index variability that we measured. It also supports the CRT index improvements being paralleled by the PSE test. The mechanistic role of ammonia is equivocal but, if anything, it is in support of our results that the group with the improvement in CRT index was the also the one in which ammonia decreased. We do, however, have a weakness due to the small sample sizes of the groups where no significant results were obtained. In these, the lack of signal might be caused by chance, although the negative results can be taken to have the power assumed in the a priori power calculation since there were only few dropouts in these groups.Our salient result was obtained in the small group of patients with an abnormal entry CRT index and randomized to active intervention. However, it was statistically significant and based on the The total number of dropouts was higher than assumed, but the sensitivity analyses still showed this did not threaten the study conclusions. The stratification after the entry CRT index did not predict dropout, but randomization to the active intervention did predict dropout. As usual, the most common complaint was bowel discomfort, but for those who chose to continue in the protocol, the complaints diminished within 7–10 days. No serious adverse event was reported.We did not observe any change in disease related quality of life in either group or strata. This contrasted with prior studies –23 and tIn summary, we showed that, in patients with cirrhosis and without manifest HE, an abnormal CRT index seemed to identify those patients who proved to cognitively normalize or improve with multimodal anti-HE intervention. Our findings needs confirmation in larger studies but indicate that the CRT test may be useful in selecting cirrhosis patients for treatment for MHE—a frequent and debilitating complication. Further, our methodological approach could be useful in a similar validation of other psychometric tests.S1 Text(PDF)Click here for additional data file.S2 Text(PDF)Click here for additional data file.S1 Data(XLS)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


The treatment with homeopathically prepared thyroxin solution (10-30) starts at the frogspawn stage. It represents a tool to learn more about the previously standardized amphibian model, where the thyroxin solution was applied from the two- legged stage on only. Lowland frogs were pretreated by immersing spawn in an aqueous molecular thyroxin dilution (10-8 parts by weight). In later stages of development (2 to 4 legged), this has been found to speed up metamorphosis by around 15%. In accordance with the homeopathic idea of detoxication or cure, hyperstimulated animals  were treated either with thyroxin that had been highly diluted and agitated in successive steps, i.e. homeopathically prepared (10-30), or analogously prepared blank solution (water). Development was monitored by documenting the number of animals that had entered the four-legged stage. It has been found that animals treated with the test solution metamorphosed more slowly than the control animals, i.e. the effect of the homeopathically prepared thyroxin was opposed to the usual effect of molecular thyroxin. The number of test animals that reached the 4- legged stage at defined points in time was slightly smaller in the group treated with homeopathically prepared thyroxin at some, but not at all points in time, compared to control. The results in this study sustain the previous multi researcher findings that highly diluted homeopathically prepared thyroxin is able to slow down metamorphosis of Rana temporaria.The influence of a highly diluted agitated, i.e. homeopathically prepared thyroxin solution (10


Arabidopsis thaliana to investigate transcriptional changes in dry seeds upon rehydration. We analyzed gene expression of dormant and after-ripened seeds of the Cvi accession over four time points and two seed compartments (the embryo and surrounding single cell layer endosperm), during the first 24 h after sowing. This work provides a global view of gene expression changes in dormant and non-dormant seeds with temporal and spatial detail, and these may be visualized via a web accessible tool (http://www.wageningenseedlab.nl/resources). A large proportion of transcripts change similarly in both dormant and non-dormant seeds upon rehydration, however, the first differences in transcript abundances become visible shortly after the initiation of imbibition, indicating that changes induced by after-ripening are detected and responded to rapidly upon rehydration. We identified several gene expression profiles which contribute to differential gene expression between dormant and non-dormant samples. Genes with enhanced expression in the endosperm of dormant seeds were overrepresented for stress-related Gene Ontology categories, suggesting a protective role for the endosperm against biotic and abiotic stress to support persistence of the dormant seed in its environment.Seed dormancy is a genetically controlled block preventing the germination of imbibed seeds in favorable conditions. It requires a period of dry storage (after-ripening) or certain environmental conditions to be overcome. Dormancy is an important seed trait, which is under selective pressure, to control the seasonal timing of seed germination. Dormant and non-dormant (after-ripened) seeds are characterized by large sets of differentially expressed genes. However, little information is available concerning the temporal and spatial transcriptional changes during early stages of rehydration in dormant and non-dormant seeds. We employed genome-wide transcriptome analysis on seeds of the model plant  Arabidopsis, seed dormancy is classified as ‘non-deep physiological dormancy’ [in common with seeds of most weeds, vegetables, and many garden flowers  and gibberellins , is of particular importance . The depArabidopsis consists of two visible events  it will proceed to germination, which in e events . First, ntenance  as seen ntenance  and, forntenance . Seed dontenance . Furtherntenance , an impontenance .Gene expression analyses on dormant and non-dormant whole seeds have been performed previously , such stArabidopsis  plants were grown in climate cell consisting of 16 h light/8 dark at a temperature of 22°C. After harvest seeds were stored in paper bags under ambient temperature and humidity. Dormancy release was followed by germination assays throughout the after-ripening period, and after 16 months dormancy was released. The imbibed seeds were dissected and the micropylar and chalazal endosperm (MCE) and the radicle and hypocotyl (RAD) were collected for RNA isolation. RNA of the RAD samples was isolated with the Absolutely RNA Nanoprep kit (Agilent Technologies) as we used earlier for our Col-0 seed samples  and these 18 samples, with three replicates for each sample, were hybridized to Affymetrix ATH1 gene chips. These 54 chips were normalized together with the 34 chips of near isogenic lines of several DELAY OF GERMINATION (DOG) loci [including samples of D and AR seeds of Ler-0 and near isogenic lines NILDOG1, NILDOG2, NILDOG3, and NILDOG6 (http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF), after removal of the control probes, 21315 genes remain. The microarray data described in this article has been deposited in the National Center for Biotechnology Information’s Gene Expression Omnibus (accession number GSE76907). The quality controls  indicate that this is a robust dataset that allows us to investigate, in seeds with opposing dormancy states, the transcriptional dynamics in two key compartments that regulate seed dormancy and germination. A web tool enabling the querying of this dataset1, under ‘Dormancy Gene Expression Plots.’This temporal and spatial sampling resulted in nine dormant and nine after-ripened samples - replacing anything less than four with four. This may slightly underestimate the number of differentially expressed genes (or their level of fold change), but helps prevent the noise region from heavily influencing the results of the fold changes; this thresholding is reflected in the numbers in Data File S1, and all numbers throughout. The gene lists were analyzed using Genetrial . Imbibed seeds were dissected into two distinct compartments, the micropylar and chalazal end of the endosperm (MCE), which acts as a barrier together with the seed coat, and the combined radicle and hypocotyl , which has to expand to break through the covering layers to complete seed germination. To capture the transcriptome dynamics, we conducted microarray analysis on RNA isolated from dry seeds and the two compartments at four time points, i.e., 3, 7, 12, and 24 hours after sowing (HAS), of both D and AR seeds . These compartments and time points were chosen to align with the sampling of germinating Col-0 seeds we performed previously  was developed in which the gene expression data of these dormancy samples were combined with the earlier mentioned seed germination samples (DELAY OF GERMINATION (NILDOG) loci, providing gene expression information concerning D and AR seeds of several genotypes with different primary dormancy levels . The expression profile of a gene of interest can be visualized across all these samples, as was previously done for anther and flower microarray datasets , and provides a useful tool for the community, allowing quick determination of the behavior of a gene of interest.A freely available web tool samples . The toodatasets . The res2 5, 59.8% of the 21,315 genes on the chip) in at least one of the 18 samples, while 7,659 genes  were expressed in all 18 samples . In dry seeds and at the early time points fewer genes were expressed, this number increased during the time course in the two compartments (MCE and RAD) and in both dormancy states . We detected nearly equal numbers of genes expressed in the MCE and the RAD  over the time course, but fewer genes were expressed in the D samples  compared to the AR samples .Over the whole time course 12,741 genes were detected to be expressed  and expressed below 5  in the other state. According to this definition, 9,735  of the genes are shared between both states whilst 40 (0.4%) are specific to D and 362 (3.3%) are specific to AR seeds. The remaining 865 transcripts (7.9%) are expressed over 6 in one state but between 5 and 6 in the other state, and are not classed as being highly specific to either physiological state. Similarly, a number of transcripts were specific to the MCE (299 genes) and the RAD (232 genes) which could be further refined (to 42 genes for the MCE and 48 for the RAD) using the transcript information of the Col-0 seeds . Subsequently, we investigated genes that were both state (D or AR) and compartment (MCE or RAD) specific, although, these resulted in very small gene sets. We identified 19 transcripts specific for the D MCE while in the D RAD only one such gene was found. Higher numbers of specifically expressed genes were found in the AR state .We identified transcript sets that were specifically expressed in either the D or the AR state, by considering genes which were expressed in one state above 6 . Like the AR Col-0 samples, the AR Cvi samples are mainly separated along the first principal component (PC), with that PC appearing to represent time. The AR Cvi samples do ‘move’ slower though, and in particular the later time points (12 and 24 HAS) lag behind those of the AR Col-0, which most likely reflects the slower germination rate compared to the AR Col-0 seeds  was 49 h for Cvi compared to 39 h for Col-0). Intriguingly, the D samples show a very different behavior; although the D samples cluster with the AR samples at 3 and 7 HAS, at later time points (12 and 24 HAS) the samples ‘stall’ around the 7 HAS position in the PCA plot . This indicates that limited transcriptional changes are occurring at the later time points in the D samples, and is supported by the number of differentially expressed genes detected between the consecutive time points in both compartments . The number of differentially expressed genes (over threefold up and down) between 3–7, 7–12, and 12–24 HAS in the AR MCE/RAD were 656/740, 614/667 and 760/558 genes, respectively. For the D samples these numbers are considerably lower, most notably between the later time points, i.e., 259/270, 45/27, and 40/30 . Over the whole time course (3–24 HAS), the total number of differentially expressed genes in the AR state is over 3000 in both tissues , which is over four times more than in the D samples .We performed a principal component analysis (PCA) to globally investigate the transcriptome time course. In our previous study of AR Col-0 seeds , the PCAFigure 4A). We identified 3194 genes which show a strong difference in expression (defined as threefold change) during the germination time course by comparing 3 and 24 HAS in D and/or AR MCE, of which a total of 1890 genes were strongly up-regulated, whereas 1304 genes were strongly down-regulated . In the second step, we tested whether these strongly differentially regulated genes are differentially expressed between D and AR MCE at the last time point . We focused on genes that are similarly expressed (an expression difference less than twofold) or differentially expressed (threefold difference) in either D or AR state. A smaller set was identified that contained genes that were neither similarly expressed nor strongly differentially expressed (showing a difference in expression between 2- and 3-fold) and these are denoted as weakly differentially expressed. Since we have selected for strongly temporally regulated genes during germination , this suggests that these gene expression profiles are similar, although less pronounced in one or the other state . Based on this analysis these temporally regulated genes were subdivided in five classes: (1) genes that are similarly expressed between D and AR MCE, (2) genes that are weakly differentially expressed in the D MCE, (3) genes that are weakly differentially expressed in the AR MCE, (4) genes that are strongly differentially expressed in D MCE, and (5) genes that are strongly differentially expressed in AR MCE .To get a more detailed view of the transcriptome similarities as well as the differences (and the gene expression profiles underlying these) in D and AR seeds we employed a two-step approach using the MCE samples. Since there is differential gene expression between D and AR seeds during the time course of imbibition, we have first focused on transcripts with a strong temporal regulation . Of this set 1174 genes  become eventually similarly expressed . In Figures 4C,D, the expression profiles of the categories identified in the second step are indicated. From the germination up-regulated gene set (643 genes), overrepresentation analysis (ORA) of gene ontology (GO) classes showed the presence of genes related to key metabolic processes related to amino acid, organic acid, nucleotide and carbohydrate as well as chromatin organization and translation, among others. The 531 down-regulated genes have functional classes related to transcription, RNA processing and stress overrepresented.We first investigated genes that are eventually expressed at a similar level  in D and AR MCE at 24 HAS. These represent transcriptional changes that occur in response to rehydration, independent from the physiological state (D or AR). In total we found 3194 genes that are strongly regulated between 3 and 24 HAS in the MCE . We subdivided the temporally strongly up- and down-regulated genes (from step 1) into those that are strongly up-regulated in the D MCE, D and AR MCE and AR MCE between 3 and 24 HAS and similar subdivision was made for the down-regulated genes . Furthermore, these sets were then subdivided based on whether the genes are strongly differentially expressed in the D or AR endosperm . These however, do not represent the complete list of genes which are differentially expressed between the D and AR states in the MCE at 24 HAS . This is explained by the presence of genes that are not strongly regulated during germination, which instead have opposing gene expression profiles between D and AR between 3 and 24 HAS, and these are also included in Figure 5.A significant fraction  of temporally regulated genes become strongly differentially expressed in either D (564 genes) or AR (773) MCE at 24 HAS . Four major gene expression profiles underlie this differential gene expression (containing at least 20 genes). The largest gene expression profile  contains genes that are actually down-regulated in AR MCE . ORA of this group identified GO functional classes related to germination/dormancy, lipid storage and stress . The second largest profile in this D set consists of 221 genes (28% of 783 genes) that are not strongly temporally regulated after imbibition (less than threefold change between 3 and 24 HAS). The strong differential expression of the D set is explained by opposing gene expression profiles between D and AR when comparing 3 and 24 HAS , with ORA showing the accumulation of stress and hormone related gene classes in D MCE. Less than 10% of the D UP genes are strongly up-regulated during germination in D but not in AR MCE (a set of 74 genes) which is overrepresented for genes whose function is related to response to abiotic and biotic stresses.A total of 783 genes are strongly differentially expressed in D MCE . The largest group  is strongly up-regulated during the time course in AR MCE, but not in D MCE ; these genes encode functions related to primary and secondary metabolism, growth and cell wall modification and are germination related. The second largest group consists of 101 genes that are strongly up-regulated (threefold) in both D and AR but are even stronger up-regulated in the AR MCE. This gene expression profile resulted in genes differentially expressed in AR MCE whose functions are related to carbohydrate and fatty acid metabolism and stress response. Interestingly, these classes overlap with those identified among the genes which became similarly expressed in AR and D MCE and those that became weakly differentially expressed in the AR MCE . Overall these analyses reveal multiple gene expression profiles that are responsible for the differential gene expression between D and AR endosperm, with ORA showing that different patterns regulate the expression of genes related to various processes.Four gene expression profiles underlie the strong activation of 824 genes in the AR MCE . In the RAD a total of 3781 genes were found to be strongly regulated (up and down) between 3 and 24 HAS (step 1), of which 39% became eventually similarly expressed in both D and AR RAD, 26% became weakly and 35% became strongly differentially expressed in either the D or AR RAD . The transcriptional differences (as well as the gene expression profiles underlying these) in D and AR RAD are shown in Supplementary Figure S2. At 24 HAS 780 genes were detected to be higher expressed in the D RAD, with the majority of them being down-regulated over our time course in the AR RAD although not in the D RAD . This is similar compared to the D MCE set, although in the MCE its relative contribution is somewhat smaller  which is explained by a relatively larger fraction of genes with opposing gene expression profiles between the D and AR state . We detected a total of 755 genes up-regulated in the AR RAD at 24 HAS, of which the largest group  was strongly up-regulated during our time course in AR RAD but not in the D RAD.The transcriptional dynamics in D and AR RAD, using this two-step approach, showed similar trends  and D RAD . The gene sets identified in the D MCE contained 7, 45, 59, and 115 genes at the time points 3, 7, 12, and 24 HAS, respectively. Using ORA of these sets identified functional classes related to abiotic and biotic stresses, protein ubiquitination and secondary metabolism . In comparison to the D MCE, relatively small gene sets (ranging between 11 and 30 genes) were identified in the D RAD and only a single overrepresented gene class was found .Finally, we employed a strategy using fold change differences to obtain gene sets that are enhanced in either D MCE or D RAD at each individual time point to investigate whether such gene sets reveal functional differences between the both compartments in the D state. The genes selected are at least threefold higher expressed in the D state and threefold higher expressed in either the MCE or the RAD. Using this strategy, we identified gene sets that are differentially expressed in D MCE . A total of over 500 genes were similarly down-regulated in expression in D and AR seed compartments. Overrepresented GO categories in both MCE and RAD included transcription and response to stress and other environmental cues. These genes likely represent remnants of the dry state or seed maturation that are repressed in rehydrated seeds regardless of their dormancy status.In our study we identified genes that are strongly differentially expressed (up or down) in both dormancy states in the time course between 3 and 24 HAS in the MCE (3194 genes found) and RAD (3781 genes found). Next, we investigated whether these genes were similarly or differentially expressed between D or AR MCE and RAD at 24 HAS. This revealed that a large proportion of those (37–39%) are expressed in a similar manner in D and AR seed compartments. Seed germination involves re-activation of, and changes in, cellular activity , with meFigure 4B; Supplementary Figure S1). Noteworthy is the finding that an overwhelming majority of the temporally down-regulated transcripts become differentially expressed in the D MCE whereas a vast majority of the temporally up-regulated genes become differentially expressed in the AR MCE with similar observations made in the RAD . Among the genes that are higher expressed in AR MCE and RAD the GO classes primary metabolic process, cellular carbohydrate metabolic process, translation, and ribosome biogenesis were overrepresented . This group of genes is activated in both D and AR seeds, but with a higher relative abundance in the AR state, likely to support the higher cellular activity and metabolic demand to support seed germination and subsequent seedling growth.The remaining genes become differentially expressed . Although we identified four major gene expression profiles for each, they show striking differences. Most genes that become higher expressed in D seeds are actually down-regulated in AR seeds (both MCE and RAD). As this down-regulation does not occur (or not to the same extent) in the D seeds, these transcripts accumulate eventually at a higher relative abundance in the D samples. This set includes genes related to seed maturation, seed dormancy, lipid storage and response to stress. Only a small proportion of the genes which are up-regulated during the time course in D seeds fail to do so in AR seeds  and both sets contain genes related to (a)biotic stress resistance. In contrast, the largest number of genes that become higher expressed in AR seeds are strongly up-regulated during the time course in AR seeds, but not (or to a lesser extent) in D seeds. Genes in this set include those involved in (cell) growth and cell wall modification, among others that are important for seed germination. In conclusion, considering all genes that become, either weakly or strongly, differentially expressed within the dormancy states, with the overwhelming majority of temporally down-regulated transcripts becoming higher in the D MCE  and the vice versa in the AR MCE , with similar proportions in the RAD. This high directional overlap emphasizes how the genes which are changing in time are changing more in the AR seeds than the D seeds.Using this transcriptome data, we set out to address three questions. The first question we aimed to answer is which profiles underlie the differential gene expression between D and AR seeds, and we described these patterns, genes and the functional classes they represent . Thus, although the level of differential gene expression is far less in D samples, the genes that do change upon rehydration are largely similar to those that change in AR samples. In conclusion, it is this huge difference in the number of transcriptional changes (both up and down) in the AR seeds in contrast to D seeds, which mainly drives differential gene expression between D and AR samples.Indeed, transcriptome changes within D seed compartments were mainly observed in a transient fashion early after sowing, with a dramatic drop thereafter resulting in a clearly reduced number of changes over the first 24 HAS in D compared to AR seed compartments. However, the genes that increase or decrease in relative abundance over the first 24 HAS in D samples are not very different since these largely overlap with genes differentially expressed in AR samples  showing that seeds respond rapidly after rehydration to the changes that occurred during after-ripening in the dry seed suggesting that after-ripening pre-sets the transcriptional response following the initiation of imbibition.After-ripening overcomes seed dormancy and allows germination in appropriate conditions . AlthougFigure 3A). It also indicates that the transcriptome of dormant seeds and that of seeds that progress toward germination start to deviate strongly between 7 and 12 HAS. Investigation of genes differentially expressed at the 7 HAS time point between D and AR samples could reveal potential transcriptional regulators that control these different transcriptional responses. Among the genes which are higher expressed (at a threefold cut-off) in both D MCE and D RAD at 7 HAS are 17 TF . The gene annotations suggest that most of these TFs are related to (a)biotic stress responses indicating that the dormant seed prepares to cope with suboptimal environmental conditions. Similarly, we looked into genes which are higher expressed in the AR MCE and AR RAD at 7 HAS and identified nine TFs (Data File S1). These TFs may include candidates that are responsible for the different gene expression responses of D and AR seeds.Although the first changes between D and AR samples are visible at 3 HAS, the transcriptomes differ more strongly at 12 and 24 HAS, indicated by the different positions of the D and AR samples in the PCA plot at these time points  that were D specific in our data set. Many dormancy up-regulated transcripts are present in dry (and early imbibed) seeds and are not (or to a lesser degree) down-regulated in D compared to AR MCE and RAD (as discussed above). This explains why so few D specific genes were detected, which was even more obvious when we looked for genes that were both D and seed compartment specific, with only 19 and one genes found for D MCE and RAD, respectively . Dormancy is regulated by the balance between two hormones ABA (required for dormancy induction) and GA  and another involved in GA degradation . This suggests that seed compartment specific expression of hormone metabolism genes contribute to the control of ABA/GA balance in dormant seeds.Lastly, we questioned whether we could identify gene sets that are both dormancy and seed compartment specifically expressed. We found very few genes . Stress related genes and proteins were previously found to be overrepresented in the endosperm of germinating Arabidopsis seeds and in its close relative cress  seed maturation (Arabidopsis mutants that have disturbed integrity of the testa or the cuticle layer are affected in seed germination, dormancy and longevity (Thus gene sets which are specific for D MCE and D RAD were identified although these were rather small. Therefore, we aimed to obtain larger gene sets which we could investigate using GO overrepresentation analysis. For this reason we employed a strategy using fold change differences to obtain gene sets that are enhanced in either D MCE or RAD at each individual time point. This resulted in larger gene sets, of which ORA identified functional classes related to abiotic and biotic stresses in the dormant endosperm (turation , which rongevity . Our resBD, RvB-V, and LB performed laboratory work; SP performed bioinformatic analysis and made the web tool; BD and SP analyzed data; BD, SP, and LB wrote the paper; MH and LB supervised the project; all authors contributed to the design of this study and all authors read, commented on and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study used data from outbreaks on four large-scale dairy farms using routine vaccination in Saudi Arabia, to evaluate the impact of vaccination and learn how to manage outbreaks more effectively in this setting. This evaluation also included an assessment of vaccine-induced neutralisation titres to the vaccine and field strains on a related farm with no history of FMD that employed an identical vaccination schedule. The incidence risk among exposed groups ranged from 2.6 to 20.1% and was significantly higher among youngstock (18.7%) compared to adults (7.4%). Evidence was found that local isolation of individual sick animals was more effective than whole group isolation and that subclinical infection and undetected circulation may occur on large-scale farms in Saudi Arabia, although both of these points require further evaluation. On the unaffected farm, the mean reciprocal titres for the vaccine and field strains were all above the cut-off supposed to correlate with clinical protection based on evidence from challenge studies. An estimate of vaccination effectiveness was not possible on the affected farms, but the incidence of FMD provides a more realistic estimation of the expected vaccine performance than in vivo studies or r1 value as it is based on field conditions and natural exposure. This study shows that analysis of field data from FMD outbreaks are a useful addition to more conventional challenge and in vitro based evaluations of vaccines and suggests further work is necessary to validate correlates of protection in field conditions.In 2015, foot-and-mouth disease (FMD) viruses of the A/ASIA/G-VII lineage emerged from the Indian sub-continent to cause outbreaks in the Middle and Near East. A factor which has been proposed to have contributed to the rapid spread of this lineage is the poor  The test outlined by the OIE 1 value). Although commonly performed, this test suffers from technical variability 1 value. Other tests that have been developed including measuring IgG subtypes and antibody avidity that may improve the predicted cross-protection, though these are less frequently used and further validation is needed in vitro and in vivo experiments to evaluate vaccines, although the results of such field studies are infrequently reported in the literature.FMD vaccines are usually evaluated either by performing challenge studies in containment facilities or by demonstrating seroconversion to antibody levels that correlate with protection in susceptible species in vitro vaccine matching performed at FAO World Reference Laboratory for FMD (WRLFMD) at The Pirbright Institute, UK have demonstrated a poor antigenic match to all commercially available vaccine strains, particularly those derived from the A/ASIA/Iran-05 viral lineage that are commonly used in the region In 2015, viruses from serotype A , previously limited to the Indian sub-continent, emerged in Saudi Arabia, Turkey, Armenia and Iran The aim of this study was to evaluate the response of routine vaccination using a polyvalent vaccine containing the A Saudi-95 strain against the A/ASIA/G-VII lineage. Data will be presented from FMD outbreaks that occurred on four large-scale dairy farms located in Saudi Arabia that were regularly using such a vaccine. As part of this investigation, sera from a different farm that did not have clinical disease but which used an identical vaccination regimen were analysed to establish titres generated using routine vaccination. Variables associated with antibody levels at the individual animal level were investigated. Data from these outbreaks will be used to improve our understanding of FMD in this type of large scale production system.22.1All dairy farms were located in a central region of Saudi Arabia. All cattle were Holstein Friesian and loose housed in dry-lots according to age and lactation status with access to outside loafing areas. Lactating cows were milked four times daily and all breeding was done by artificial insemination. Both youngstock and adults were located on the same units but managed separately so considered separately. Electronic individual animal records were kept according to a unique ear tag identification including disease events. Replacement stock were either sourced from the same farm or introduced from a limited number of other farms in the same area. All bull calves were sold by around 14 days of age.2.2Data from FMD outbreaks on four different farms were used for this study. The outbreaks occurred between 3rd September 2015 and 3rd April 2017 and were the first reported outbreaks of FMD A/ASIA/G-VII in Saudi Arabia. Upon suspicion of an outbreak, farms notified the relevant national authorities and samples were submitted to the WRLFMD for confirmation, RNA sequencing and strain characterisation (vaccine matching). Farms instigated varying degrees of internal isolation depending on the facilities available. The date and unique identification number of animals with FMD were recorded manually and entered onto the farm management software. Animals were recorded as a case of FMD if the animal was seen salivating with any of the following clinical signs: mouth lesions, feet lesions, teat lesions, fever, reduced feed intake, and lameness. All recording was done by farm staff supervised by veterinary surgeons employed by the farms.All farms in the study regularly used a polyvalent, killed, aqueous adjuvanted , NSP purified FMDV vaccine . The vaccine contained the following FMD virus strains: O Manisa, O-3039, A Iran-05, A Saudi-95, Asia-1 Shamir and SAT-2. A four dose primary course was given to youngstock at a target of four, five, six and seven months of age. This schedule was based on the recommendations outlined by Kitching and Salt 2.3In order to assess the antibody titres generated from this vaccine, a fifth farm was purposively selected that had no recent history of clinical FMD but used the same vaccine type (Aftovaxpur) and schedule. The last reported FMD outbreak on this farm was in 2008 before any of the animals currently on the farm were born. Animals may have been introduced from other farms, but were not included in the sampling. To ensure even age representation, an age-stratified sampling scheme was used. A target of 15 cattle were randomly selected for sampling from each of the following age groups: 6–12 months, 1–2 years, 2–3 years, 3–4 years, >4 years. A single group with animals of the appropriate age group was selected for sampling. Individuals were selected within each group based on the order they arrived in the handling facility. For the animals in the 6–12 month age strata, the farm had already sent samples to the WRLFMD on their own accord to monitor post-vaccination titres. Therefore, these samples were used rather that implementing re-sampling. The samples were taken between October 2014 and January 2015. All other animals were sampled in January 2016. The vaccine type and schedules that were used in cattle sampled earlier were identical so this was not considered likely to influence the results.The farm supplied data for each animal including: individual unique identification number, date of birth, sampling date, parity, days in milk, pregnancy status, days in calf, days until next calving, days post vaccination, number of lifetime doses of vaccine received and vaccination dates.2.4All serum samples were tested for non-structural protein (NSP) and structural protein (SP) antibodies. NSP antibodies were measured using a PrioCHECK™ FMD NS Antibody kit  with a value greater than or equal to 50% percent inhibition being positive. All samples were run in duplicate and results designated as inconclusive if the values were either side of the positive cut-off and excluded from subsequent analysis. SP antibody titres were measured using Virus Neutralisation Tests (VNT) as described previously 2.5The size of the affected groups on the study farms ranged from 3800 to 23,200 heads . Due to Logistic regression was used to compare the odds of being NSP antibody positive in the different age categories. Interval regression was used to analyse the VNT data, as this method allows a value to fall within defined intervals and also accounts for censoring of data when titres are below (left censoring) or above (right censoring) the thresholds of detection. To illustrate the variability, the true result is expected to lay between dilution series either side of the reported value. For example a result of 1/16 is expected to lie between 1/8 and 1/32. These boundaries are highlighted in 33.1Data were analysed from four farms that had clinical FMD confirmed by the WRLFMD as lineage A/ASIA/G-VII. The four farms are labelled as A, B, C and D in order of the onset date for the index case. Outbreak data are summarised in The epidemic curves are shown in Maps of affected groups on each farm are shown in Farms isolated animals to varying degrees depending on the facilities available. All isolation units were located on the same farm site. For Farms A and C, isolation tended to be close to where the cases were identified in neighbouring pens. Farm A initially isolated individual sick adults within an isolation unit around 1 km away from the main cattle houses. After approximately ten cases were isolated, this changed to a house within the main farm area. After around 60–70 cases had occurred, animals were left within their original groups. Farm C isolated individual sick youngstock in a pen close to the affected groups. Farms B and D performed more extensive isolation. The latter moved all clinically affected adults to an isolation unit 6–8 km away. Farm B moved six groups of animals  located close to the index case to the isolation units immediately upon disease suspicion. Due to the outbreak progressing, 19 days after the index case, a row of 11 youngstock corals  were moved to the same isolation facility around 3 km away. Once in the isolation unit, diseased animals were separated from non-diseased leading to a restructuring of the groups such that epidemic curves for individual groups cannot be reliably created. Due to pressure over animal numbers, restocking of ten empty pens took place around three weeks later with cases subsequently occurring in these groups 20 days after restocking.3.2For each farm, the index case occurred between 15 and 65 days (mean 33.8 days) since the last herd level vaccination . RegardiThere was no apparent correlation between the incidence risk and time since vaccination prior to the index case . On FarmAll four farms implemented reactive vaccination once the index case had been observed. The timings and types of vaccinations relative to the index case are presented in 3.3Data on the animals sampled for serology from an unaffected farm are presented in 10 reciprocal titres for A Iran-05, A Saudi-95 and A/SAU/2/2015 were 2.84 , 2.87  and 2.48 , respectively. The non-overlapping confidence intervals indicate that the heterologous titres to the latter field strain were significantly lower than for the two homologous vaccine strains. However, all individual titres and their lower 95% confidence limits were above the 2.067 protection cut-off reported by Barnett et al. Each sample was tested for virus neutralisation titres to the two homologous strains of serotype A in the vaccine  and a field strain isolated from an affected large-scale farm in Saudi Arabia (A/SAU/2/2015). Based on interval regression, the mean logUnivariable interval regression analysis is shown in 4in vitro studies are heavily relied upon for evaluating FMD vaccines. The PD50 or PPG measurements are based on challenge experiments with a homologous virus In vitro vaccine matching is based on a comparison of neutralisation titres of post vaccination sera to the field and homologous vaccine strains. An r1-value of 0.3 or above is used to indicate a vaccine that should provide sufficient match to confer protection 50, a vaccine may afford protection even if the r1-value is less than 0.3 50 and r1-value as they are derived from field conditions with natural exposure to FMDV.Detailed descriptions of disease outbreaks in endemic settings are rarely reported in the literature, but can provide useful information on the effectiveness of control measures that could be applied in endemic and FMD free settings. Challenge and 50 of each component was >6.0, and vaccination was carried out every 105 days after a four dose primary course. Although FMD outbreaks occurred, experience from incursions into this type of production system suggests the observed incidence was lower than would be expected in an immunologically naïve herd, despite the poor in vitro antigenic matching result This study presented data from four FMD outbreaks due to the viral lineage A/ASIA/G-VII that occurred on large-scale dairy farms employing frequent, regular vaccination. The vaccination schedule employed a multi-valent vaccine where the PDEmpirical data relating these experimental parameters to actual FMD outbreaks in the field are lacking, with only a few examples in the literature from Turkey The serological study on an unaffected farm using the same vaccination protocols had the aim of providing evidence of the likely titres on the affected farms prior to the outbreak. The relatively high proportion of NSP positive cattle in the 1–<2 year age group was surprising. NSP antibodies may be present due to: FMD virus infection; maternally derived antibody prior to waning at approximately six months of age; use of non-NSP purified vaccines; or repeated use of a purified vaccine that may contain trace amounts of NSP. No clinical FMD of any serotype had been reported on this farm for several years prior to the birth of these NSP positive animals. All of the tested animals were born on the farm and were of sufficient age not to have maternally derived antibodies. It is unlikely that these results indicate antibody responses to trace amounts of NSP in the vaccine, as a linear trend in percent positivity would be expected with age (correlating correlates with the lifetime number of doses received). On this basis, the high proportion of NSP positive animals in the 1–2 year age group may indicate that subclinical or mild infection had been present in this group. The VNT levels to the serotype A strains tested in this study were not different in NSP seropositive animals indicating the exposure was likely due to another serotype. Although cattle were NSP antibody positive in this group, further groups would need to have been sampled to assess the full extent of NSP seropositivity among this age cohort on this farm. However, direct contact was possible between neighbouring groups and equipment and personnel were shared making intergroup transmission likely. The observed virus neutralising titres were all greater than a protective cut-off suggested by Barnett et al. Examination of the epidemic curves on the farms revealed two distinct phases consistent with reports from the farm staff. On two farms these phases were separated by short gaps (8 and 9 days; These outbreaks also provided some insights on the potential impact of isolation on the progression on an outbreak within an affected farm. On all four farms, isolation was implemented early during the outbreak and to varying degrees depending on the resources available. On two farms, local isolation of individual animals seemed to be effective in containing the spread of virus. NSP serology may have assisted with this interpretation by providing evidence of infection in non-clinically affected groups. However, the occurrence of regular outbreaks and the presence of maternal antibody would have made interpretation difficult. Outbreaks on the other two farms were more widespread despite extensive isolation of whole groups of animals. This may indicate that the movement of whole groups of animals may present some risk of transmission within the herd and that the procedures should be modified. However, these farms were also larger in size which may have also been a factor related to the wider outbreak distribution. Further observations of field outbreaks will provide further evidence of optimal management of outbreaks to reduce the impact of incursions.In summary, analysis of data from these outbreaks provided useful information on the potential effectiveness of different isolation procedures for limiting spread of virus and also the A Saudi-95 vaccine for protecting against lineage A/ASIA/G-VII strains. There is also evidence that the usual serological correlate of protection may need further investigation to assess its relevance in different field conditions.


Supplemental Digital Content is available in the text Skin cancer screening has undoubted potential to reduce cancer-specific morbidity and mortality. Total-body exams remain the prevailing concept of skin cancer screening even if effectiveness and value of this method are controversial. Meanwhile, store and forward teledermatology was shown to be a reliable instrument for several diagnostic purposes mostly in specialized dermatology settings. The objective of this study was to evaluate most convenient mobile teledermatology interventions as instruments for skin cancer screening in a representative population.Prospective diagnostic study with visitors of a skin cancer screening campaign in Switzerland. Histopathology was used as reference standard. Mobile teledermatology with or without dermoscopic images was assessed for performance as a screening test . Outcomes were sensitivity, specificity, and predictive values.Seven cases of skin cancer were present among 195 skin lesions. All skin cancers were ruled-in by teledermatology with or without dermoscopic images . The addition of dermoscopic images to conventional images resulted in higher specificity (85% vs. 77%), allowing reduction of unnecessary further testing in a larger proportion of skin lesions.Store and forward mobile teledermatology could serve as an instrument for population-based skin cancer screening because of favorable test performance. Moreover, the effectiveness of skin cancer screening in general still remains to be determined. So far, the prevailing concept of skin cancer screening is the total-body exam executed by trained physicians. This type of intervention, however, is very expensive and possibly disproportional to introduce on a population-based scale. The nationwide skin cancer screening program in Germany, for instance, relied on total-body exams but surprisingly failed to result in improvements of skin cancer mortality rates despite promising results from a pilot project.,8 The role of total-body exams might be the one of a case-finding procedure in an at-risk population, rather than a population-based screening intervention. In contrast, lesion directed examinations are more time-efficient and have similar detection rates, but still require time-expensive face-to-face consultations and similar constraints as to total-body examinations apply. Teledermatological assessments on the other hand, especially lesion directed store and forward procedures can be performed in a most speedy manner. In several former studies, the reliability of teledermatology based management recommendations was shown to be comparable to face-to-face consultations.–15 Moreover, recent technical advancements led to mobile phone assisted dermoscopy even enabling “mobile teledermoscopy,” combining high technical standards of teledermatological image acquisition with mobile phone convenience. Feasibility and reliability for cancer detection with such mobile teledermoscopy has already been shown in high prevalence settings.,17Skin cancer is on the rise globally and especially so in Switzerland. Once these issues are resolved, image acquisition could be transferred to settings outside medical practices and broader population based screening could be implemented.Given time-efficiency, reliability, and the widespread dissemination of powerful digital cameras integrated in mobile phones, the remaining technical limitations to mobile teledermatology for skin cancer screening are few and may lie in standardization of the specific methods of image acquisition to achieve highest possible reliability.To assess a screening intervention in this context, test performance measures  in directing malignant lesions to further evaluation (and treatment) and withholding these measures in non-malignant skin lesions are needed to know. Also, the detection rate of malignant skin lesions  in a representative low-prevalence setting is relevant to estimate the value of the screening intervention. So far, however, only little research in mobile teledermatology has been conducted in settings representative for population based screening, and research concentrated on specialized secondary or tertiary care settings. Furthermore, the additional value of teledermoscopy on top of conventional teledermatology for screening interventions is still unclear.Therefore, the aim of this study was to estimate the diagnostic performance  of lesion-directed mobile teledermatology with or without dermoscopic imaging as a skin cancer screening intervention in a population representing a low-prevalence setting.22.1We conducted a prospective diagnostic study with data collection during a public skin cancer screening campaign at the Department of Dermatology at the University Hospital of Zürich in May 2013. Ethics committee of the Canton of Zürich approved the study on May 8, 2013 (KEK-ZH-Nr. 2013–0119). The study was conducted according to the tenants of the declaration of Helsinki and to good clinical practice standards.2.2The “National skin cancer day” is a yearly conducted skin cancer screening campaign in Switzerland and is carried out in dermatology clinics and hospitals since 2006. Anyone interested may visit a participating site without scheduled appointment to present specific skin lesions to be checked for malignancy . In order to reduce administrative expenditures during the campaign, participants do not register at the clinic but are guided through the screening anonymously without any information being documented. For the purpose of this study and to evaluate quality of the procedures during the campaign, we prospectively documented skin lesions with digital images and structured clinical information and followed outcomes of biopsies.At the reception, participants were given consecutive numbers in order of their appearance in the clinic. Participants were then allocated to 1 of 18 examination rooms. In 8 of the 18 examination rooms, personnel and camera equipment for study participation was available and participants were approached for study participation. The allocation to the 18 examination rooms was performed consecutively in order of appearance at the reception and according to availability in next possible examination room. Thereby, we sought to prevent selection of specific skin lesions for specific examination rooms. We included skin lesions of participants that provided informed consent and the documentation of the skin lesion with macroscopic and microscopic digital images succeeded.2.3The lesion-directed clinical examinations were performed by residents of the Department of Dermatology with an on-call backup by senior staff for assistance in uncertain cases. From examinations, the following data were filled in a standardized form: age, sex, personal or family history of skin cancer, skin type according to Fitzpatrick, the estimated total number of moles (>100 or <100), suspected diagnoses and management recommendations for the skin lesion under question.When skin cancer was suspected, participants were offered appointments (immediately or within a few days) to remove the skin lesions or to perform biopsy. The biopsy results were available to us and served as the reference standard for skin cancer being present or not. When skin cancer was ruled out clinically, obviously no additional biopsy was performed and the clinical decision served as reference standard.2.42.4.1 All acquired images were JPEGs with a resolution of 2592 × 1936 pixels.Medicine students who completed a 1-hour training workshop in skin lesion photography acquired 3 images of every skin lesion utilizing the integrated 5-megapixel camera of an iPod (Apple Inc.) touch fifth generation. First, an overview image that showed anatomical region of the lesion, second a macroscopic image from approximately 10 cm that showed the surrounding skin including the dispersion of other skin lesions, third a dermoscopic image was taken using the Handyscope  .2.4.2The images were arranged by skin lesions and organized in 2 packages, the first package including macroscopic images only and the second package including the additional teledermoscopic images. The packages were stored on encrypted hard-drives and sequentially forwarded to the teledermatologist (see acknowledgment) with a 1-month washout time interval between the first and the second package. The teledermatologist was a board certified dermatologist and very experienced in the teledermatological examination of skin lesions including digital teledermoscopy. Along with the images, the teledermatologist was given the information about the patients’ age, sex, history of skin cancer, Fitzpatrick skin type, and the estimated number of moles retrieved from the questionnaire of the clinical examination. The teledermatologist was aware of her task being skin cancer screening but was left unaware of the aim of the study and blinded towards suspected diagnoses and management recommendations already given by the dermatologists at the screening campaign and towards biopsy results. For every skin lesion, the teledermatologist first decided whether the provided images were adequate to allow assessment for cancer screening at all. Secondly, in adequate cases, management recommendations had to be given in 3 categories: (a) no follow-up required, (b) clinical assessment required, (c) direct excision or biopsy.2.5Quantitative variables are reported as medians and interquartile ranges, categorical variables are reported as counts and proportions. Management recommendations from teledermatology were dichotomized as follows: “no follow-up required” was categorized as negative outcome of the screening test and “clinical assessment required” or “direct excision or biopsy” was categorized as positive test outcome. Skin lesions that had histopathology-confirmed skin cancers were categorized as cancer-positive, all others as cancer-negative.κ). All analyses were performed in duplicate for the 2 test methods with or without teledermoscopic images.We calculated the number of patients to be screened to detect a case of skin cancer, by dividing the total number of histology confirmed skin cancer by the number participants included in the study. Furthermore, we calculated test sensitivity, specificity, and predictive values in according to the test outcomes categorized above with reference to cancer being present or not according to the biopsy confirmed results. Agreement between teledermatology and gold standard are expressed by concordance rate as well as Cohen's Kappa  were counted at the screening campaign on May 13th, 2013 and 225 skin lesions were seen in examination rooms equipped for study participation. In 13 (5.8%) of these informed consent was unavailable and in 17 (7.6%) no digital images were acquired because of technical problems, thus resulting in 195 (86.7%) of the eligible skin lesions included in the study  of the cases, in the image package including teledermoscopic images 192 (98.5%) skin lesions were suitable. To detect 1 case of skin cancer, we needed to screen 27 skin lesions in our sample.3.3κ = 0.20).Test performance measures of conventional teledermatology were 100% sensitivity, 76.6% specificity, 14.6% positive predictive value, and 100% negative predictive value. Conventional teledermatology was able to rule-out the need for any further follow-up in 134 of 182 skin lesions (73.6%) without missing any of the skin cancers in the sample. Overall concordance of conventional teledermatology with reference standard was 77.5% (κ = 0.29).Additional teledermoscopy on top of conventional teledermatology performed with 100% sensitivity, 84.9% specificity, 20.0% positive predictive value, and 100% negative predictive value. The rule-out of any further follow-up was possible in 157 of 192 (81.8%) skin lesions without missing any of the skin cancers in the sample. Overall concordance of teledermatology featuring teledermoscopy with reference standard was 85.4% .3.4In order to assess whether different levels of clinical presentations were associated with different test-performance, we applied a binomial logistic regression model with the dependent variable being the agreement of the teledermatology with reference standard. As independent variables, we added all clinical variables from the clinical assessment.For teledermatology without teledermoscopic images we found no association of clinical presentations with test performance. In teledermatology including teledermoscopy, however, the clinical presentations of atypical mole and age were both associated with lesser odds for agreement with reference standard: atypical mole odds ratio for agreement (OR) = 0.05 ; age OR = 0.96 (95% CI = 0.92–0.99).4In this study, mobile teledermatology detected all cases of skin cancer in a low prevalence population and accurately ruled out the necessity of further testing in 3 out of 4 cases. Mobile teledermoscopy in addition to conventional images increased specificity of the screening allowing correct rule-out in 4 out of 5 skin lesions. Mobile teledermoscopy also had higher practicability featuring a substantially lower rate of images classified as inadequate for screening compared with conventional mobile teledermatology.,17 In a very similar setting as ours, a high management concordance between face-to-face consultations and conventional teledermatology has recently been found and cancer screening utilizing teledermatology was suggested. Here, we followed the diagnostic chain beyond agreement with clinical management recommendations (suffering from interrater variability) and linked the results to a valid gold-standard represented by histopathology, therefore allowing test performance measurements in a screening population.Findings from our study are in line with previous research demonstrating very high cancer detection rates of mobile teledermoscopy in high-prevalence settings.The specific methods in skin cancer screening are still to debate and our results emphasize teledermatology's potential contribution to this field: first, sensitivity and negative predictive value for ruling in skin cancers  was 100% clearly arguing for qualification of the technique as a screening intervention, because no case of skin cancer was falsely ruled out. Furthermore, only minimal training of photographers was needed to enable acquisition of images suitable for the teledermatologist arguing for practicability of the technique. We did not systematically assess the teledermatologist's time expenditures to perform the assessments; however, each set  was finished within “one afternoon” strongly arguing for time-efficiency of the procedure considering that the image material was derived from face-to-face exams performed by 8 physicians working an entire day each. Looking at the additional value of teledermoscopy on top of conventional teledermatology, we found that more skin lesions were suitable to be screened but more importantly, specificity increased allowing more lesions to be ruled-out by the screening with the exception of atypical moles. Thus, considering that additional expenses in technical equipment and time to perform 1 additional image are small, mobile teledermoscopy might be a valuable enhancement in addition to conventional mobile teledermatology because it can effectively decrease unnecessary follow-up exams due to false-positive screening outcomes. Similar procedures are by now only proposed and integrated in remote and medically under-supported areas but such a service might also decrease skin cancer rates in other healthcare settings when offered to the broad population. Even if total-body exams presumably have higher detection rate in 1 specific individual, the population-wide detection rate may be higher using a teledermoscopy service. This because of lower barriers and easier access to teledermatology than to total-body exams and because of already very high acceptance among the population. Notably, such a screening would ultimately depend on self-detection of skin lesions by individuals among the population. The comparably high cancer rate we found in our population speaks in favor of the population's ability for self-detection, furthermore this ability has specifically been found before in a comparable screening setting.Our results show how teledermatology could contribute to skin cancer screening by image acquisition also outside medical practice by minimally instructed personnel (possibly even patients themselves) as piloted for other clinical questions in dermatology.4.1The main strength and novelty of our study is the head-to-head comparison of teledermatological skin cancer screening interventions in a representative population with a valid gold-standard available. Limitations to this study are the following: first, only 1 teledermatologist was involved and since there is no standardized training or generally accepted method of image acquisition and image evaluation, reproducibility of our results is uncertain. The low-level of efforts needed to implement image acquisition combined with the positive results per se, however, are encouraging for further pursuit of the concept. Secondly, teledermatology may still have missed skin cancers because histopathology was not available in every 195 skin lesion assessed, however, these hypothetical skin cancers would have also been missed by the face-to-face examinations. Therefore, teledermatology was at least non-inferior to face-to-face examinations that are themselves the commonly accepted standard in skin cancer screening.4.2For further research we suggest that teledermatology's test performance is investigated depending on reproducible standards of image acquisition and image evaluation using skin lesions originating from a population representative for skin cancer screening. Especially, there might still be room left to improve specificity of the screening without renouncing sensitivity that appears already to be very high. Further steps then include research in delivery and design of a population-based teledermatology screening intervention  before a population wide reduction of skin cancer burden can be attempted.We conclude that store and forward mobile teledermatology is inexpensive, practicable, and able to produce highly accurate management recommendations in the specific role of skin cancer screening. Population-based skin cancer prevention programs could consider implementing mobile teledermatology services as screening tests rather than directly offering total-body exams.