Equine grass sickness is associated with major abnormalities in the ultrastructure of skeletal neuromuscular junctions.

BACKGROUND: Equine grass sickness (EGS) is a frequently fatal multisystem neuropathy of equids. The aetiology is unknown; proposed causes include toxicoinfection with Clostridium botulinum and a mycotoxicosis. The effect of EGS on the organisation and structural integrity of the skeletal neuromuscular junction (NMJ), the target of botulinum neurotoxins (BoNTs), is unknown. OBJECTIVES: To compare the organisation and structural integrity of skeletal NMJs from EGS horses, control horses and one horse with a presumptive diagnosis of botulism. STUDY DESIGN: Blinded, retrospective case control. METHODS: NMJs in samples of diaphragm or intercostal muscle from six EGS horses, three control horses and one equine botulism case were compared using electron microscopy, morphometry and confocal light microscopy. RESULTS: A significantly higher percentage of EGS NMJs had abnormal morphology (EGS 72.2%, 95% CI 55.6-84.4; Controls 6.9%, 1.7-23.8; OR 35.1, 8.47-244.8; p < 0.001). EGS NMJs had a significantly lower mean volume fraction occupied by synaptic vesicles (SVs) (EGS 18.7%, 12.6-28.0; Controls 36.3%, 20.8-63.4; p = 0.024). EGS NMJs had evidence of accelerated SV exocytosis and SV depletion, accumulation of neurofilament-like material in terminal boutons and/or bouton degeneration. NMJs from the botulism horse had dense packing of SVs towards the presynaptic membrane active zone, consistent with BoNT intoxication, but had absence of the abnormalities identified in EGS NMJs. MAIN LIMITATIONS: Group sizes were limited by difficulties obtaining suitably processed samples. Ages of control and EGS horses differed. Botulism was diagnosed based on clinical and post mortem findings. CONCLUSIONS: EGS is associated with major changes in skeletal NMJ ultrastructure that are inconsistent with the effects of BoNTs. SV depletion may reflect increased exocytosis coupled with reduced repopulation of SVs via anterograde axonal transport and endocytosis, consistent with the action of an excitatory presynaptic toxin and/or neurotransmitter reuptake inhibitor. Skeletal NMJs represent a previously unrecognised target for the toxin that causes EGS.

The effect of season, management and endocrinopathies on vitamin D status in horses.

BACKGROUND: Vitamin D deficiency is common in humans and is increasingly linked to the pathogenesis of a multitude of diseases including obesity and metabolic syndrome. The biology of vitamin D in horses is poorly described; the relative contribution of the diet and skin synthesis to circulating concentrations is unclear and associations with the endocrine disease have not been explored. OBJECTIVES: To determine the relationship between management, season and endocrine disease and vitamin D status in horses. STUDY DESIGN: Cross-sectional cohort study. METHODS: Plasma concentrations of 25-hydroxyvitamin D2 (25(OH)D2 ) and D3 (25(OH)D3 ) were measured by liquid chromatography-tandem mass spectrometry in 34 healthy unsupplemented grazing ponies and 22 stabled Thoroughbreds receiving supplementary vitamin D3 in feed. A nested group of 18 grazing ponies were sampled on long and short days (>12 and <12 h of light/day) to determine the effect of sunlight exposure. In addition, the relationships between age, sex, adiposity, serum insulin, adrenocorticotropic hormone and vitamin D status were assessed in a mixed group of 107 horses using a linear regression model. RESULTS: All animals had a measurable level of 25(OH)D2 (median 10.7 nmol/L) whilst 25(OH)D3 was only detected in Thoroughbreds receiving D3 supplementation. Thoroughbreds had lower concentrations of 25(OH)D2 than ponies (7.4 vs. 12.6 nmol/L, p < 0.01). In grazing ponies, 25(OH)D2 concentrations were significantly higher on long days compared to short days (14.4 vs. 8.7 nmol/L, p < 0.01), whilst 25(OH)D3 was undetectable. Measures of increased adiposity, but not basal insulin, were associated with higher 25(OH)D2 concentrations, conversely to humans. Increasing ACTH was associated with lower 25(OH)D2 (p < 0.01). MAIN LIMITATIONS: Vitamin D2 concentrations were not measured in grass or forage. CONCLUSIONS: In horses 25(OH)D2 is the predominant vitamin D metabolite, and there is an apparent lack of endogenous vitamin D3 production. The relationship between vitamin D and endocrine disorders in horses does not reflect that of other species and warrants further investigation.

Real-time three-dimensional echocardiography for left atrial volume assessment in Thoroughbred racehorses: Observer variability and comparison with two-dimensional echocardiography.

BACKGROUND: Left atrial size predicts cardiac morbidity and mortality in humans and dogs. Real-time three-dimensional echocardiography (3DE) may be reliable for assessing left atrial volume (LAV) in horses. OBJECTIVES: To determine intra- and interobserver variability estimates of 3DE-LAV and compare it to that of 2DE-LAV estimates. STUDY DESIGN: Method comparison. METHODS: 3DE datasets were obtained from 40 horses, then graded for quality, creating a final study population of 22 horses. The 3DE and 2DE maximum LAV (LAVmax ) and minimum LAV (LAVmin ) were measured, and left atrial emptying volume (LA EV) and left atrial ejection fraction (LA EF) were calculated, from the same 3D dataset on four occasions using (a) a semi-automatic surface recognition algorithm and (b) a modified Simpson's method of discs. 3DE LAV measurements were repeated by a second observer. RESULTS: For 3DE, median LAVmax was 596cm3 for observer one, and 852 cm3 for observer two, LAVmin was 373 cm3 for observer one and 533 cm3 for observer two. Low intraobserver measurement variation was observed for LAVmax and LAVmin , with horse-level intraclass correlation coefficients (ICChorse ) for both observers between 76% and 85% (horse added as random effect). The interobserver ICC was 58% for LAVmax and 50% for LAVmin on averaged measurements (with observer added as random effect), indicating consistent differences between observers. While intraobserver variation was similar for 2DE LAVmax measurements, it was greater for LAVmin (ICChorse  = 67%). The intermethod ICC for 3DE vs 2DE was low at 14% for LAVmax and ~0% for LAVmin , indicating less-consistent differences with method. MAIN LIMITATIONS: Small study population, low observer number, use of different imaging modalities (fundamental frequency and octave harmonics). CONCLUSIONS: 3DE assessment of LAV was reliable, suggesting suitability for longitudinal evaluation of clinical cases. Clinicians should be aware of differences in LAV measurements between observers. More defined measurement guidelines may improve repeatability.

Equine grass sickness (a multiple systems neuropathy) is associated with alterations in the gastrointestinal mycobiome.

BACKGROUND: Equine grass sickness (EGS) is a multiple systems neuropathy of grazing horses of unknown aetiology. An apparently identical disease occurs in cats, dogs, rabbits, hares, sheep, alpacas and llamas. Many of the risk factors for EGS are consistent with it being a pasture mycotoxicosis. To identify potential causal fungi, the gastrointestinal mycobiota of EGS horses were evaluated using targeted amplicon sequencing, and compared with those of two control groups. Samples were collected post mortem from up to 5 sites in the gastrointestinal tracts of EGS horses (EGS group; 150 samples from 54 horses) and from control horses that were not grazing EGS pastures and that had been euthanased for reasons other than neurologic and gastrointestinal diseases (CTRL group; 67 samples from 31 horses). Faecal samples were also collected from healthy control horses that were co-grazing pastures with EGS horses at disease onset (CoG group; 48 samples from 48 horses). RESULTS: Mycobiota at all 5 gastrointestinal sites comprised large numbers of fungi exhibiting diverse taxonomy, growth morphology, trophic mode and ecological guild. FUNGuild analysis parsed most phylotypes as ingested environmental microfungi, agaricoids and yeasts, with only 1% as gastrointestinal adapted animal endosymbionts. Mycobiota richness varied throughout the gastrointestinal tract and was greater in EGS horses. There were significant inter-group and inter-site differences in mycobiota structure. A large number of phylotypes were differentially abundant among groups. Key phylotypes (n = 56) associated with EGS were identified that had high abundance and high prevalence in EGS samples, significantly increased abundance in EGS samples, and were important determinants of the inter-group differences in mycobiota structure. Many key phylotypes were extremophiles and/or were predicted to produce cytotoxic and/or neurotoxic extrolites. CONCLUSIONS: This is the first reported molecular characterisation of the gastrointestinal mycobiota of grazing horses. Key phylotypes associated with EGS were identified. Further work is required to determine whether neurotoxic extrolites from key phylotypes contribute to EGS aetiology or whether the association of key phylotypes and EGS is a consequence of disease or is non-causal.

Incorporation of Equine Learning Theory into the Undergraduate Curriculum.

Working as an equine veterinarian carries a high risk of occupational injury, with the behavior of the horse frequently reported as a cause for these injuries. Risk of injury is one reason cited by undergraduate veterinary students that would prevent them from entering large animal practice, and newly graduated veterinarians have been shown to be at increased risk of sustaining an occupational injury compared with more experienced colleagues. A cohort of pre-final-year undergraduate veterinary students were given a 45-minute lecture on learning theory and its application in equine practice, completing a questionnaire before (pre) and after (immediately [post] and several weeks [delayed post]) to investigate whether receiving a single lecture alters undergraduate veterinary students' perception of dealing with difficult horses in equine practice. The undergraduate veterinary students' attitudes to the behavior scenarios altered from the pre-questionnaire to the post- and delayed post-questionnaires. They were less likely to choose more traditional methods of restraining or controlling the horse (such as a twitch) and more likely to choose an option based on learning theory after the lecture. They also reported that if they had to deal with one of these scenarios in practice following the lecture, they would feel more confident, more likely to succeed in completing the intervention, and less likely to be injured. This study suggests that an educational intervention can help change the attitudes and confidence of undergraduate students when working with difficult horses.

Pathological bradyarrhythmia in horses.

Pathological bradyarrhythmia is rare in horses but should be especially considered when presented with a horse that has signs consistent with episodic weakness or collapse. This paper reviews the literature describing our current knowledge of, and possible mechanisms causing, clinically significant bradyarrhythmia in horses.

Inducing weight loss in native ponies: is straw a viable alternative to hay?

BACKGROUND: Obesity is a growing problem in UK equine population. Achieving weight loss in obese horses and ponies at risk of laminitis is an important but often challenging objective. METHODS: We hypothesised that supplementing poor winter pasture with a mix of barley straw and hay (50:50) rather than hay alone (group B) would lead to weight loss in grazing equids over winter. For this purpose, a group of 40 horses were fed either the straw mix (group A) or hay alone (group B) over winter. RESULTS: Over the study period, all animals in group A (n=25) lost weight with a mean weight change of -27±17 kg, while in group B (n=15) only 3 out of 15 lost weight (20 per cent), and overall, group B gained weight (+6±18 kg). CONCLUSIONS: This study suggests that straw is a cost-effective and low-energy roughage, which may be a useful alternative to hay alone when trying to induce weight loss in grazing equids over winter. There were no episodes of colic or laminitis during the study period in either group.

Examination of Horses with Cardiac Disease.

Despite advances, increased convenience, and availability of echocardiography and other diagnostic techniques in equine cardiology, a comprehensive history and clinical examination still forms the essential first step in any cardiac evaluation. This article summarizes the approach to the cardiac examination at rest, highlighting key areas for the clinician to assess, and stressing the importance of context for assessing the significance of any abnormalities detected. Ancillary techniques, such as blood pressure measurement and the laboratory assessment of cardiac disease in the horse, are also introduced.

Dysregulation of Cortisol Metabolism in Equine Pituitary Pars Intermedia Dysfunction.

Equine Cushing disease [pituitary pars intermedia dysfunction (PPID)] is a common condition of older horses, but its pathophysiology is complex and poorly understood. In contrast to pituitary-dependent hyperadrenocorticism in other species, PPID is characterized by elevated plasma ACTH but not elevated plasma cortisol. In this study, we address this paradox and the hypothesis that PPID is a syndrome of ACTH excess in which there is dysregulation of peripheral glucocorticoid metabolism and binding. In 14 horses with PPID compared with 15 healthy controls, we show that in plasma, cortisol levels and cortisol binding to corticosteroid binding globulin were not different; in urine, glucocorticoid and androgen metabolites were increased up to fourfold; in liver, 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) expression was reduced; in perirenal adipose tissue, 11ß-HSD1 and carbonyl reductase 1 expression was increased; and tissue cortisol levels were not measurably different. The combination of normal plasma cortisol with markedly enhanced urinary cortisol metabolite excretion and dysregulated tissue-specific steroid-metabolizing enzymes suggests that cortisol clearance is increased in horses with PPID. We infer that the ACTH excess may be compensatory and pituitary pathology and autonomous secretion may be a secondary rather than primary pathology. It is possible that successful therapy in PPID may be targeted either at lowering ACTH or, paradoxically, at reducing cortisol clearance.

Species-specific regulation of angiogenesis by glucocorticoids reveals contrasting effects on inflammatory and angiogenic pathways.

Glucocorticoids are potent inhibitors of angiogenesis in the rodent in vivo and in vitro but the mechanism by which this occurs has not been determined. Administration of glucocorticoids is used to treat a number of conditions in horses but the angiogenic response of equine vessels to glucocorticoids and, therefore, the potential role of glucocorticoids in pathogenesis and treatment of equine disease, is unknown. This study addressed the hypothesis that glucocorticoids would be angiostatic both in equine and murine blood vessels.The mouse aortic ring model of angiogenesis was adapted to assess the effects of cortisol in equine vessels. Vessel rings were cultured under basal conditions or exposed to: foetal bovine serum (FBS; 3%); cortisol (600 nM), cortisol (600nM) plus FBS (3%), cortisol (600nM) plus either the glucocorticoid receptor antagonist RU486 or the mineralocorticoid receptor antagonist spironolactone. In murine aortae cortisol inhibited and FBS stimulated new vessel growth. In contrast, in equine blood vessels FBS alone had no effect but cortisol alone, or in combination with FBS, dramatically increased new vessel growth compared with controls. This effect was blocked by glucocorticoid receptor antagonism but not by mineralocorticoid antagonism. The transcriptomes of murine and equine angiogenesis demonstrated cortisol-induced down-regulation of inflammatory pathways in both species but up-regulation of pro-angiogenic pathways selectively in the horse. Genes up-regulated in the horse and down-regulated in mice were associated with the extracellular matrix. These data call into question our understanding of glucocorticoids as angiostatic in every species and may be of clinical relevance in the horse.

Assessment of tissue-specific cortisol activity with regard to degeneration of the suspensory ligaments in horses with pituitary pars intermedia dysfunction.

OBJECTIVE To identify signs of tissue-specific cortisol activity in samples of suspensory ligament (SL) and neck skin tissue from horses with and without pituitary pars intermedia dysfunction (PPID). SAMPLE Suspensory ligament and neck skin tissue samples obtained from 26 euthanized horses with and without PPID. PROCEDURES Tissue samples were collected from 12 horses with and 14 horses without PPID (controls). Two control horses had received treatment with dexamethasone; data from those horses were not used in statistical analyses. The other 12 control horses were classified as old horses (≥ 14 years old) and young horses (≤ 9 years old). Standard histologic staining, staining for proteoglycan accumulation, and immunostaining of SL and neck skin tissue sections for glucocorticoid receptors, insulin, 11ß hydroxysteroid dehydrogenase type 1, and 11ß hydroxysteroid dehydrogenase type 2 were performed. Findings for horses with PPID were compared with findings for young and old horses without PPID. RESULTS Compared with findings for old and young control horses, there were significantly more cells stained for glucocorticoid receptors in SL samples and for 11 ß hydroxysteroid dehydrogenase type 1 in SL and skin tissue samples from horses with PPID. Insulin could not be detected in any of the SL or skin tissue samples. Horses with PPID had evidence of SL degeneration with significantly increased proteoglycan accumulation. Neck skin tissue was found to be significantly thinner in PPID-affected horses than in young control horses. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that tissue-specific dysregulation of cortisol metabolism may contribute to the SL degeneration associated with PPID in horses.

Utilization of Computer-Aided Detection for Digital Screening Mammography in the United States, 2008 to 2016.

PURPOSE: Computer-aided detection (CAD) for screening mammography is a software technology designed to improve radiologists' reading performance. Since 2007, multiple Breast Cancer Surveillance Consortium research papers have shown that CAD decreases performance by increasing recalls and decreasing the detection of invasive cancer while increasing the detection of ductal carcinoma in situ. The aim of this study was to test the hypothesis that CAD use by digital mammography facilities would decrease over time. METHODS: In August 2007, August 2011, and March 2016, the FDA database of certified mammography facilities was accessed, and a random sample of 400 of approximately 8,500 total facilities was generated. In 2008 and 2011, a telephone survey was conducted of the facilities regarding digital mammography and CAD use. In 2016, facility websites were reviewed before calling the facilities. Bonferroni-corrected P values were used to assess statistical differences in the proportion of CAD at digital facilities for the three surveys. RESULTS: The mean proportion of digital facilities using CAD was 91.4%, including 91.4% (128 of 140) in 2008, 90.2% (238 of 264) in 2011, and 92.3% (358 of 388) in 2016. The difference for 2008 versus 2011 was 1.3% (95% confidence interval [CI], -0.5% to 7.7%), for 2011 versus 2016 was -2.1% (95% CI, -6.9% to 2.7%), and for 2008 versus 2016 was -0.8% (95% CI, -6.7% to 5.0%). CONCLUSIONS: In three national surveys, it was found that CAD use at US digital screening mammography facilities was stable from 2008 to 2016. This persistent utilization is relevant to the debate on the value of targeting ductal carcinoma in situ in screening.

Carbonyl reductase 1 catalyzes 20ß-reduction of glucocorticoids, modulating receptor activation and metabolic complications of obesity.

Carbonyl Reductase 1 (CBR1) is a ubiquitously expressed cytosolic enzyme important in exogenous drug metabolism but the physiological function of which is unknown. Here, we describe a role for CBR1 in metabolism of glucocorticoids. CBR1 catalyzes the NADPH- dependent production of 20ß-dihydrocortisol (20ß-DHF) from cortisol. CBR1 provides the major route of cortisol metabolism in horses and is up-regulated in adipose tissue in obesity in horses, humans and mice. We demonstrate that 20ß-DHF is a weak endogenous agonist of the human glucocorticoid receptor (GR). Pharmacological inhibition of CBR1 in diet-induced obesity in mice results in more marked glucose intolerance with evidence for enhanced hepatic GR signaling. These findings suggest that CBR1 generating 20ß-dihydrocortisol is a novel pathway modulating GR activation and providing enzymatic protection against excessive GR activation in obesity.

Vascular Dysfunction in Horses with Endocrinopathic Laminitis.

Endocrinopathic laminitis (EL) is a vascular condition of the equine hoof resulting in severe lameness with both welfare and economic implications. EL occurs in association with equine metabolic syndrome and equine Cushing's disease. Vascular dysfunction, most commonly due to endothelial dysfunction, is associated with cardiovascular risk in people with metabolic syndrome and Cushing's syndrome. We tested the hypothesis that horses with EL have vascular, specifically endothelial, dysfunction. Healthy horses (n = 6) and horses with EL (n = 6) destined for euthanasia were recruited. We studied vessels from the hooves (laminar artery, laminar vein) and the facial skin (facial skin arteries) by small vessel wire myography. The response to vasoconstrictors phenylephrine (10-9-10-5M) and 5-hydroxytryptamine (5HT; 10-9-10-5M) and the vasodilator acetylcholine (10-9-10-5M) was determined. In comparison with healthy controls, acetylcholine-induced relaxation was dramatically reduced in all intact vessels from horses with EL (% relaxation of healthy laminar arteries 323.5 ± 94.1% v EL 90.8 ± 4.4%, P = 0.01, laminar veins 129.4 ± 14.8% v EL 71.2 ± 4.1%, P = 0.005 and facial skin arteries 182.0 ± 40.7% v EL 91.4 ± 4.5%, P = 0.01). In addition, contractile responses to phenylephrine and 5HT were increased in intact laminar veins from horses with EL compared with healthy horses; these differences were endothelium-independent. Sensitivity to phenylephrine was reduced in intact laminar arteries (P = 0.006) and veins (P = 0.009) from horses with EL. Horses with EL exhibit significant vascular dysfunction in laminar vessels and in facial skin arteries. The systemic nature of the abnormalities suggest this dysfunction is associated with the underlying endocrinopathy and not local changes to the hoof.

Four Principles to Consider Before Advising Women on Screening Mammography.

This article reviews four important screening principles applicable to screening mammography in order to facilitate informed choice. The first principle is that screening may help, hurt, or have no effect. In order to reduce mortality and mastectomy rates, screening must reduce the rate of advanced disease, which likely has not happened. Through overdiagnosis, screening produces substantial harm by increasing both lumpectomy and mastectomy rates, which offsets the often-promised benefit of less invasive therapy. Next, all-cause mortality is the most reliable way to measure the efficacy of a screening intervention. Disease-specific mortality is biased due to difficulties in attribution of cause of death and to increased mortality due to overdiagnosis and the resulting overtreatment with radiotherapy and chemotherapy. To enhance participation, the benefit from screening is often presented in relative instead of absolute terms. Third, some screening statistics must be interpreted with caution. Increased survival time and the percentage of early-stage tumors at detection sound plausible, but are affected by lead-time and length biases. In addition, analyses that only include women who attend screening cannot reliably correct for selection bias. The final principle is that accounting for tumor biology is important for accurate estimates of lead time, and the potential benefit from screening. Since "early detection" is actually late in a tumor's lifetime, the time window when screen detection might extend a woman's life is narrow, as many tumors that can form metastases will already have done so. Instead of encouraging screening mammography, physicians should help women make an informed decision as with any medical intervention.