Rapid Isothermal Detection of Pathogenic Clostridioides difficile Using Recombinase Polymerase Amplification.

Nosocomial-associated diarrhea due to Clostridioides difficile infection (CDI) is diagnosed after sample precultivation by the detection of the toxins in enzyme immunoassays or via toxin gene nucleic acid amplification. Rapid and direct diagnosis is important for targeted treatment to prevent severe cases and recurrence. We developed two singleplex and a one-pot duplex fluorescent 15 min isothermal recombinase polymerase amplification (RPA) assays targeting the toxin genes A and B (tcdA and tcdB). Furthermore, we adapted the singleplex RPA to a 3D-printed microreactor device. Analytical sensitivity was determined using a DNA standard and DNA extracts of 20 C. difficile strains with different toxinotypes. Nineteen clostridial and gastrointestinal bacteria strains were used to determine analytical specificity. Adaptation of singleplex assays to duplex assays in a 50 µL volume required optimized primer and probe concentrations. A volume reduction by one-fourth (12.4 µL) was established for the 3D-printed microreactor. Mixing of RPA was confirmed as essential for optimal analytical sensitivity. Detection limits (LOD) ranging from 119 to 1411 DNA molecules detected were similar in the duplex tube format and in the singleplex 3D-printed microreactor format. The duplex RPA allows the simultaneous detection of both toxins important for the timely and reliable diagnosis of CDI. The 3D-printed reaction chamber can be developed into a microfluidic lab-on-a-chip system use at the point of care.

Comparing lying behaviour of young riding horses on days in an individual indoor box, on an outdoor paddock alone, or in pairs and in the following night.

BACKGROUND: Horses must lie down to go into vital rapid-eye movement (REM) sleep. If they are not lying down for sufficiently long periods they can become so sleep-deprived that they collapse uncontrollably, which results in a risk of injuries. OBJECTIVES: To investigate how recumbency as a prerequisite to REM sleep on the experimental days and the following nights was influenced by changes in social and spatial environment throughout the day. STUDY DESIGN: Cross-over design in which subjects experienced each experimental condition twice. METHODS: Observations were conducted on a horse farm. Ten young horses in training were observed on days when they were alone indoors in a box, on an outdoor paddock alone, on the same paddock in pairs, and in the following night stabled alone. The number of lying bouts and the total lying duration throughout the day and night were assessed automatically using 3D-accelerometers and data were evaluated using mixed-models. RESULTS: Horses had a higher number of lying bouts during the days (p = 0.05, by a factor of 1.21 [95% CI: 1.00-1.45]) and longer lying duration at night (p < 0.001, by a factor of 11.25 [6.47-18.40]). On average, the number of lying bouts and the duration of lying increased from being indoors alone, to being outdoors alone, and outdoors in pairs although this could not be statistically supported (bouts: p = 0.5, by a factor of 1.08 [0.84-1.36] and 1.17 [0.91-1.48]; p = 0.6, duration: by a factor of 1.39 [0.73-2.93] and 1.38 [0.68-2.78]). MAIN LIMITATIONS: A small number of horses were observed and there was large variability between days within horses. CONCLUSIONS: We found some indications that open space and a social companion throughout the day increased time lying down in the day as well as during the following nights thus allowing for more REM sleep.

Social Box: A New Housing System Increases Social Interactions among Stallions.

In domestic conditions, adult stallions are mostly housed individually in internal stables to reduce the risk of injuries during social interactions. Social deprivation in horses results in physiological stress and behavioural problems. The aim of this study was to test the "social box" (SB), which allows closer physical contact between neighbouring horses. Eight pairs of stallions (n = 16) were filmed over a 24 h period in the SB and in their usual box stables, "conventional boxes" (CB), which strongly restrict tactile contact. The effect of housing in the SB on behaviour and the occurrence and characteristics of injuries was investigated. The total duration of active social interactions was significantly higher in the SB than in the CB (51.1 vs. 4.9 min, p < 0.0001). Positive interactions accounted for about 71% of the total duration of interactions in SB and CB stabling. The stallions interacted significantly more often in the SB than in the CB (113.5 vs. 23.8 social interaction sequences over 24 h, p < 0.0001). No grievous injuries were recorded. The social box appears to be a suitable solution to give adult stallions the possibility of having physical interactions. Therefore, it can be considered a substantial environmental enrichment for singly housed horses.

Social Box: Influence of a New Housing System on the Social Interactions of Stallions When Driven in Pairs.

In order to improve the housing conditions of stallions in individual boxes, we tested a so-called "social box" allowing increased physical contact between neighbouring horses. This study investigated whether housing stallions in social boxes changes the number of social interactions during carriage driving. We hypothesised that the stay in social boxes would decrease the number of unwanted social interactions between stallions when driven in pairs. Eight Franches-Montagnes breeding stallions were observed when driven in pairs with a "neutral" stallion housed in a so-called "conventional box", strongly limiting physical contact. They were driven on a standardised route over the course of four days before, during, and after being housed in social boxes. The type and frequency of behaviours of the pairs and the interventions of the groom and the driver during the test drives were assessed live and using video recordings. Results from linear mixed-effect models show that unwanted social interactions decreased during and after the stallions were housed in the social box (p < 0.001). Stallions' interactions also decreased over the four days (p < 0.01), suggesting a habituation to the test conditions by learning not to interact, or by subtly settling dominance. The social box tended to decrease unwanted social behaviours of stallions driven in pairs and could therefore be used as an environmental enrichment for horses.

More Than 500 Kids Could Be Saved Each Year! Ten Consensus Actions to Improve Quality of Pediatric Resuscitation in DACH-Countries (Austria, Germany, and Switzerland).

• Quality and outcome of pediatric resuscitation often does not achieve recommended goals. • Quality improvement initiatives with the aim of better survival rates and decreased morbidity of resuscitated children are urgently needed. • These initiatives should include an action framework for a comprehensive, fundamental, and interprofessional reorientation of clinical and organizational structures concerning resuscitation and post-resuscitation care of children. • The authors of this DACH position statement suggest the implementation of 10 evidence-based actions (for out-of-hospital and in-house cardiac arrests) that should improve survival rates and decrease morbidity of resuscitated children with better neurological outcome and quality of life.

Schnellnachweis von SARS-CoV-2 mit recombinase polymerase amplification.

The COVID-19 pandemic highlights the need for fast and simple assays for nucleic acid detection. As an isothermal alternative to RT-qPCR, we outline the development of a detection scheme for SARS-CoV-2 RNA based on reverse transcription recombinase polymerase amplification (RT-RPA) technology. RPA uses recombination proteins in combination with a DNA polymerase for rapid amplification of target DNA at a constant temperature (39-42 °C) within 10 to 20 minutes and can be monitored in real-time with fluorescent probes.

3D Printed Monolithic Microreactors for Real-Time Detection of Klebsiella pneumoniae and the Resistance Gene blaNDM-1 by Recombinase Polymerase Amplification.

We investigate the compatibility of three 3D printing materials towards real-time recombinase polymerase amplification (rtRPA). Both the general ability of the rtRPA reaction to occur while in contact with the cured 3D printing materials as well as the residual autofluorescence and fluorescence drift in dependence on post curing of the materials is characterized. We 3D printed monolithic rtRPA microreactors and subjected the devices to different post curing protocols. Residual autofluorescence and drift, as well as rtRPA kinetics, were then measured in a custom-made mobile temperature-controlled fluorescence reader (mTFR). Furthermore, we investigated the effects of storage on the devices over a 30-day period. Finally, we present the single- and duplex rtRPA detection of both the organism-specific Klebsiella haemolysin (khe) gene and the New Delhi metallo-ß-lactamase 1 (blaNDM-1) gene from Klebsiella pneumoniae. Results: No combination of 3D printing resin and post curing protocol completely inhibited the rtRPA reaction. The autofluorescence and fluorescence drift measured were found to be highly dependent on printing material and wavelength. Storage had the effect of decreasing the autofluorescence of the investigated materials. Both khe and blaNDM-1 were successfully detected by single- and duplex-rtRPA inside monolithic rtRPA microreactors printed from NextDent Ortho Clear (NXOC). The reaction kinetics were found to be close to those observed for rtRPA performed in a microcentrifuge tube without the need for mixing during amplification. Singleplex assays for both khe and blaNDM-1 achieved a limit of detection of 2.5 × 101 DNA copies while the duplex assay achieved 2.5 × 101 DNA copies for khe and 2.5 × 102 DNA copies for blaNDM-1. Impact: We expand on the state of the art by demonstrating a technology that can manufacture monolithic microfluidic devices that are readily suitable for rtRPA. The devices exhibit very low autofluorescence and fluorescence drift and are compatible with RPA chemistry without the need for any surface pre-treatment such as blocking with, e.g., BSA or PEG.

Rapid Detection of SARS-CoV-2 by Low Volume Real-Time Single Tube Reverse Transcription Recombinase Polymerase Amplification Using an Exo Probe with an Internally Linked Quencher (Exo-IQ).

BACKGROUND: The current outbreak of SARS-CoV-2 has spread to almost every country with more than 5 million confirmed cases and over 300,000 deaths as of May 26, 2020. Rapid first-line testing protocols are needed for outbreak control and surveillance. METHODS: We used computational and manual designs to generate a suitable set of reverse transcription recombinase polymerase amplification (RT-RPA) primer and exonuclease probe, internally quenched (exo-IQ), sequences targeting the SARS-CoV-2 N gene. RT-RPA sensitivity was determined by amplification of in vitro transcribed RNA standards. Assay selectivity was demonstrated with a selectivity panel of 32 nucleic acid samples derived from common respiratory viruses. To validate the assay against full-length SARS-CoV-2 RNA, total viral RNA derived from cell culture supernatant and 19 nasopharyngeal swab samples (8 positive and 11 negative for SARS-CoV-2) were screened. All results were compared to established RT-qPCR assays. RESULTS: The 95% detection probability of the RT-RPA assay was determined to be 7.74 (95% CI: 2.87-27.39) RNA copies per reaction. The assay showed no cross-reactivity to any other screened coronaviruses or respiratory viruses of clinical significance. The developed RT-RPA assay produced 100% diagnostic sensitivity and specificity when compared to RT-qPCR (n = 20). CONCLUSIONS: With a run time of 15 to 20 minutes and first results being available in under 7 minutes for high RNA concentrations, the reported assay constitutes one of the fastest nucleic acid based detection methods for SARS-CoV-2 to date and may provide a simple-to-use alternative to RT-qPCR for first-line screening at the point of need.

Frameshift Variant in MFSD12 Explains the Mushroom Coat Color Dilution in Shetland Ponies.

Mushroom is a unique coat color phenotype in Shetland Ponies characterized by the dilution of the chestnut coat color to a sepia tone and is hypothesized to be a recessive trait. A genome wide association study (GWAS), utilizing the Affymetrix 670K array (MNEc670k) and a single locus mixed linear model analysis (EMMAX), identified a locus on ECA7 for further investigation (Pcorrected = 2.08 × 10-10). This locus contained a 3 Mb run of homozygosity in the 12 mushroom ponies tested. Analysis of high throughput Illumina sequencing data from one mushroom Shetland pony compared to 87 genomes from horses of various breeds, uncovered a frameshift variant, p.Asp201fs, in the MFSD12 gene encoding the major facilitator superfamily domain containing 12 protein. This variant was perfectly concordant with phenotype in 96 Shetland Ponies (P = 1.15 × 10-22), was identified in the closely related Miniature Horse for which the mushroom phenotype is suspected to occur (fmu = 0.02), and was absent in 252 individuals from seven additional breeds not reported to have the mushroom phenotype. MFSD12 is highly expressed in melanocytes and variants in this gene in humans, mice, and dogs impact pigmentation. Given the role of MFSD12 in melanogenesis, we propose that p.Asp201fs is causal for the dilution observed in mushroom ponies.

Elevated Sensitivity to Tactile Stimuli in Stereotypic Horses.

Although stereotypic behaviors are a common problem in captive animals, why certain individuals are more prone to develop them remains elusive. In horses, individuals show considerable differences in how they perceive and react to external events, suggesting that this may partially account for the emergence of stereotypies in this species. In this study, we focused on crib-biting, the most common stereotypy displayed by horses. We compared how established crib-biters ("CB" = 19) and normal controls ("C" = 18) differed in response to a standard "personality" assessment test battery, i.e., reactivity to humans, tactile sensitivity, social reactivity, locomotor activity, and curiosity vs. fearfulness (both in novel and suddenness situations). Our analyses showed that crib-biters only differed from control horses in their tactile sensitivity, suggesting an elevated sensitivity to tactile stimuli. We suggest that this higher tactile sensitivity could be due to altered dopamine or endogenous opioid physiology, resulting from chronic stress exposition. We discuss these findings in relation to the hypothesis that there may be a genetic predisposition for stereotypic behavior in horses, and in relation to current animal husbandry and management practices.

Stereotypic horses (Equus caballus) are not cognitively impaired.

Stereotypies in animals are thought to arise from an interaction between genetic predisposition and sub-optimal housing conditions. In domestic horses, a well-studied stereotypy is crib-biting, an abnormal behaviour that appears to help individuals to cope with stressful situations. One prominent hypothesis states that animals affected by stereotypies are cognitively less flexible compared to healthy controls, due to sensitization of a specific brain area, the basal ganglia. The aim of this study was to test this hypothesis in crib-biting and healthy controls, using a cognitive task, reversal learning, which has been used as a diagnostic for basal ganglia dysfunction. The procedure consisted of exposing subjects to four learning tasks; first and second acquisition, and their reversals. For each task, we measured the number of trials to reach criterion and heart rate and heart-rate variability. Importantly, we did not try to prevent crib-biters from executing their stereotypic behaviour. We found that the first reversal learning task required the largest number of trials, confirming its challenging nature. Interestingly, the second reversal learning task required significantly fewer trials to reach criterion, suggesting generalisation learning. However, we did not find any performance differences across groups; both stereotypic and control animals required a similar numbers of trials and did not differ in their physiological responses. Our results thus challenge the widely held belief that crib-biting horses, and stereotypic animals more generally, are cognitively impaired. We conclude that cognitive underperformance may occur in stereotypic horses if they are prevented from crib-biting to cope with experienced stress.

Social information in equine movement gestalts.

One model of signal evolution is based on the notion that behaviours become increasingly detached from their original biological functions to obtain a communicative value. Selection may not always favour the evolution of such transitions, for instance, if signalling is costly due to predators usurping signal production. Here, we collected inertial movement sensing data recorded from multiple locations in free-ranging horses (Equus caballus), which we subjected to a machine learning algorithm to extract kinematic gestalt profiles. This yielded surprisingly rich and multi-layered sets of information. In particular, we were able to discriminate identity, breed, sex and some personality traits from the overall movement patterns of freely moving subjects. Our study suggests that, by attending to movement gestalts, domestic horses, and probably many other group-living animals, have access to rich social information passively but reliably made available by conspecifics, a finding that we discuss in relation with current signal evolution theories.

Erratum: Segregation of information about emotional arousal and valence in horse whinnies.

This corrects the article DOI: 10.1038/srep09989.

Space Allowance of the Littered Area Affects Lying Behavior in Group-Housed Horses.

Horses can sleep while standing; however, recumbency is required for rapid eye movement (REM) sleep and therefore essential. Previous research indicated a minimal duration of recumbency of 30 min per 24 h to perform a minimal duration of REM sleep. For group-housed horses, suitable lying area represents a potentially limited resource. In Switzerland, minimal dimensions for the space allowance of the littered area are therefore legally required. To assess the effect of different space allowances of the littered area on lying behavior, 38 horses in 8 groups were exposed to 4 treatments for 11 days each; T0: no litter provided, T0.5: 0.5× minimal dimensions, T1: minimal dimensions, and T1.5: 1.5× minimal dimensions. Non-littered areas were covered with hard rubber mats. Lying behavior was observed during the last 72 h of each treatment. The total number of lying bouts per 24 h was similar in treatments providing litter, whereas in treatment T0, recumbency occurred only rarely (F1,93 = 14.74, p = 0.0002) with the majority of horses lying down for less than 30 min per 24 h ([Formula: see text], p = 0.0006). Overall, the total duration of recumbency per 24 h increased with increasing dimensions of the littered area, whereby the effect attenuated between treatment T1 and T1.5 in high-ranking horses but continued in low-ranking horses (F1,91 = 3.22, p = 0.076). Furthermore, low-ranking horses showed considerably more forcedly terminated lying bouts in treatments T0.5 and T1, but were similar to high-ranking horses in T1.5 (F1,76 = 8.43, p = 0.005). Nonetheless, a number of individuals showed durations of recumbency of less than 30 min per 24 h even in treatment T1.5. The lying behavior was dependent on the availability of a soft and deformable surface for recumbency. A beneficial effect of enlarged dimensions of the littered area was shown by increased durations of recumbency and decreased proportion of forcedly terminated lying bouts in low-ranking horses. Taking this into account, it became evident that the minimal dimensions for the littered area as implemented in the Swiss animal welfare legislation do not ensure undisturbed lying behavior for all members of a given group.

Toward a Choice-Based Judgment Bias Task for Horses.

Judgment bias tasks for nonhuman animals are promising tools to assess emotional valence as a measure of animal welfare. In view of establishing a valid judgment bias task for horses, the present study aimed to evaluate 2 versions (go/no-go and active choice) of an auditory judgment bias task for horses in terms of acquisition learning and discrimination of ambiguous cues. Five mares and 5 stallions were randomly assigned to the 2 designs and trained for 10 trials per day to acquire different operant responses to a low-frequency tone and a high-frequency tone, respectively. Following acquisition learning, horses were tested on 4 days with 3 ambiguous-tone trials interspersed between the 10 high-tone and low-tone trials. All 5 go/no-go horses but only one active-choice horse successfully learned their task, indicating that it is more difficult to train horses on an active choice task than on a go/no-go task. During testing, however, go/no-go horses did not differentiate between the 3 different ambiguous cues, thereby making the validity of the test results questionable in terms of emotional valence.

Perception of emotional valence in horse whinnies.

BACKGROUND: Non-human animals often produce different types of vocalisations in negative and positive contexts (i.e. different valence), similar to humans, in which crying is associated with negative emotions and laughter is associated with positive ones. However, some types of vocalisations (e.g. contact calls, human speech) can be produced in both negative and positive contexts, and changes in valence are only accompanied by slight structural differences. Although such acoustically graded signals associated with opposite valence have been highlighted in some species, it is not known if conspecifics discriminate them, and if contagion of emotional valence occurs as a result. We tested whether domestic horses perceive, and are affected by, the emotional valence of whinnies produced by both familiar and unfamiliar conspecifics. We measured physiological and behavioural reactions to whinnies recorded during emotionally negative (social separation) and positive (social reunion) situations. RESULTS: We show that horses perceive acoustic cues to both valence and familiarity present in whinnies. They reacted differently (respiration rate, head movements, height of the head and latency to respond) to separation and reunion whinnies when produced by familiar, but not unfamiliar individuals. They were also more emotionally aroused (shorter inter-pulse intervals and higher locomotion) when hearing unfamiliar compared to familiar whinnies. In addition, the acoustic parameters of separation and reunion whinnies affected the physiology and behaviour of conspecifics in a continuous way. However, we did not find clear evidence for contagion of emotional valence. CONCLUSIONS: Horses are thus able to perceive changes linked to emotional valence within a given vocalisation type, similar to perception of affective prosody in humans. Whinnies produced in either separation or reunion situations seem to constitute acoustically graded variants with distinct functions, enabling horses to increase their apparent vocal repertoire size.

A Nonsense Variant in the ST14 Gene in Akhal-Teke Horses with Naked Foal Syndrome.

Naked foal syndrome (NFS) is a genodermatosis in the Akhal-Teke horse breed. We provide the first scientific description of this phenotype. Affected horses have almost no hair and show a mild ichthyosis. So far, all known NFS affected horses died between a few weeks and 3 yr of age. It is not clear whether a specific pathology caused the premature deaths. NFS is inherited as a monogenic autosomal recessive trait. We mapped the disease causing genetic variant to two segments on chromosomes 7 and 27 in the equine genome. Whole genome sequencing of two affected horses, two obligate carriers, and 75 control horses from other breeds revealed a single nonsynonymous genetic variant on the chromosome 7 segment that was perfectly associated with NFS. The affected horses were homozygous for ST14:c.388G>T, a nonsense variant that truncates >80% of the open reading frame of the ST14 gene (p.Glu130*). The variant leads to partial nonsense-mediated decay of the mutant transcript. Genetic variants in the ST14 gene are responsible for autosomal recessive congenital ichthyosis 11 in humans. Thus, the identified equine ST14:c.388G>T variant is an excellent candidate causative variant for NFS, and the affected horses represent a large animal model for a known human genodermatosis. Our findings will enable genetic testing to avoid the nonintentional breeding of NFS-affected foals.

Are Eyes a Mirror of the Soul? What Eye Wrinkles Reveal about a Horse's Emotional State.

Finding valid indicators of emotional states is one of the biggest challenges in animal welfare science. Here, we investigated in horses whether variation in the expression of eye wrinkles caused by contraction of the inner eyebrow raiser reflects emotional valence. By confronting horses with positive and negative conditions, we aimed to induce positive and negative emotional states, hypothesising that positive emotions would reduce whereas negative emotions would increase eye wrinkle expression. Sixteen horses were individually exposed in a balanced order to two positive (grooming, food anticipation) and two negative conditions (food competition, waving a plastic bag). Each condition lasted for 60 seconds and was preceded by a 60 second control phase. Throughout both phases, pictures of the eyes were taken, and for each horse four pictures per condition and phase were randomly selected. Pictures were scored in random order and by two experimenters blind to condition and phase for six outcome measures: qualitative impression, eyelid shape, markedness of the wrinkles, presence of eye white, number of wrinkles, and the angle between the line through the eyeball and the highest wrinkle. The angle decreased during grooming and increased during food competition compared to control phases, whereas the two phases did not differ during food anticipation and the plastic bag condition. No effects on the other outcome measures were detected. Taken together, we have defined a set of measures to assess eye wrinkle expression reliably, of which one measure was affected by the conditions the horses were exposed to. Variation in eye wrinkle expression might provide valuable information on horse welfare but further validation of specific measures across different conditions is needed.

A Non-Synonymous HMGA2 Variant Decreases Height in Shetland Ponies and Other Small Horses.

The identification of quantitative trait loci (QTL) such as height and their underlying causative variants is still challenging and often requires large sample sizes. In humans hundreds of loci with small effects control the heritable portion of height variability. In domestic animals, typically only a few loci with comparatively large effects explain a major fraction of the heritability. We investigated height at withers in Shetland ponies and mapped a QTL to ECA 6 by genome-wide association (GWAS) using a small cohort of only 48 animals and the Illumina equine SNP70 BeadChip. Fine-mapping revealed a shared haplotype block of 793 kb in small Shetland ponies. The HMGA2 gene, known to be associated with height in horses and many other species, was located in the associated haplotype. After closing a gap in the equine reference genome we identified a non-synonymous variant in the first exon of HMGA2 in small Shetland ponies. The variant was predicted to affect the functionally important first AT-hook DNA binding domain of the HMGA2 protein (c.83G>A; p.G28E). We assessed the functional impact and found impaired DNA binding of a peptide with the mutant sequence in an electrophoretic mobility shift assay. This suggests that the HMGA2 variant also affects DNA binding in vivo and thus leads to reduced growth and a smaller stature in Shetland ponies. The identified HMGA2 variant also segregates in several other pony breeds but was not found in regular-sized horse breeds. We therefore conclude that we identified a quantitative trait nucleotide for height in horses.