Genomic loci associated with performance limiting equine overriding spinous processes (kissing spines).

Commonly known as "Kissing Spines" (KS), the pathological mechanisms underlying impingement and overriding of spinous processes (ORSPs) in horses are poorly understood. Thoroughbreds, Warmbloods, and stock-type breeds, including Paint Horses and Quarter Horses are at increased risk for developing clinical signs of KS. A total of 155 stock-type and Warmblood horses presented at collaborating veterinary clinics and hospitals were examined using a strict clinical and radiographical phenotyping scheme to grade each horse from 0 for unaffected controls to 4 for severe KS. Following genotyping with the Illumina Equine SNP70 array (Illumina, Inc.) a Genome Wide Association Study (GWAS) using 61,229 filtered individual Single Nucleotide Polymorphisms (SNPs) was performed to the KS grade phenotype. Two significantly associated SNPs (BIEC2-668062 and BIEC2-668013) on chromosome 25 defined a ~1.4 Gb candidate region containing approximately 17 coding genes (EquCab3) and 195 ENSEMBL annotated variants. Investigation of the best associated SNP (BIEC2-668062) on chr25 demonstrates a significant correlation with an increase in one KS grade, on average, per A allele in this population. A significant effect of breed group, age, height or sex was not observed in this population. These preliminary results demonstrate the potential for KS diagnosis and preventative measures for WB/ST individuals supported by increased genetic risk for more severe KS grade. We propose further research including other affected breeds and evaluating causative variants, as well as the effect of BIEC2-668062 in these populations.

Evaluation of dynamic testing for pituitary pars intermedia dysfunction diagnosis in donkeys.

BACKGROUND: Endocrine disorders are common in donkeys. Pituitary pars intermedia dysfunction (PPID) is thought to be a frequent disturbance in donkeys due to their longevity. However, information on PPID dynamic testing in donkeys is lacking. OBJECTIVES: The objective of this study was to evaluate the previously described guidelines for PPID diagnosis in horses in donkeys with suspicion of PPID. STUDY DESIGN: Prospective experimental study. METHODS: Eighty donkeys were evaluated for PPID suspicion based on clinical signs and baseline adrenocorticotropic hormone (ACTH) concentrations. Six mix-breed donkeys (one jack and five non-pregnant jennies) fulfilling inclusion criteria were subjected to dexamethasone suppression test (DST), thyrotropin-releasing hormone stimulation test (TRH) and combined DST-TRH challenge. Tests were interpreted according to guidelines for PPID diagnosis in horses. RESULTS: Donkeys fulfilling inclusion criteria were diagnosed with PPID by TRH stimulation test (six of six). Both DST (three of six) and DST-TRH (4/6) challenges failed to detect those animals and showed conflicting results. Similarly, cortisol basal concentrations were not consistent with PPID suspicion. MAIN LIMITATIONS: Characterisation of seasonal and geographical location effect on baseline ACTH concentrations and response to TRH is compelling in this species. Further studies with a larger number of donkeys are needed. CONCLUSIONS: This is the first study in donkeys to evaluate common dynamic tests used for PPID diagnosis in horses. Preliminary results agree with the guidelines for PPID diagnosis in horses and baseline ACTH measurement followed by TRH challenge are recommended tests for diagnosis of PPID in donkeys.

Streptococcus equi Infections in Horses: Guidelines for Treatment, Control, and Prevention of Strangles-Revised Consensus Statement.

This consensus statement update reflects our current published knowledge and opinion about clinical signs, pathogenesis, epidemiology, treatment, complications, and control of strangles. This updated statement emphasizes varying presentations in the context of existing underlying immunity and carrier states of strangles in the transmission of disease. The statement redefines the "gold standard" for detection of possible infection and reviews the new technologies available in polymerase chain reaction diagnosis and serology and their use in outbreak control and prevention. We reiterate the importance of judicious use of antibiotics in horses with strangles. This updated consensus statement reviews current vaccine technology and the importance of linking vaccination with currently advocated disease control and prevention programs to facilitate the eradication of endemic infections while safely maintaining herd immunity. Differentiation between immune responses to primary and repeated exposure of subclinically infected animals and responses induced by vaccination is also addressed.

A large-scale, rapid public health response to rabies in an organ recipient and the previously undiagnosed organ donor.

This article describes and contrasts the public health response to two human rabies cases: one organ recipient diagnosed within days of symptom onset and the transplant donor who was diagnosed 18 months post-symptom onset. In response to an organ-transplant-related rabies case diagnosed in 2013, organ donor and recipient investigations were conducted by multiple public health agencies. Persons with potential exposure to infectious patient materials were assessed for rabies virus exposure. An exposure investigation was conducted to determine the source of the organ donor's infection. Over 100 persons from more than 20 agencies spent over 2700 h conducting contact investigations in healthcare, military and community settings. The 564 persons assessed include 417 healthcare workers [5.8% recommended for post-exposure prophylaxis (PEP)], 96 community contacts (15.6% recommended for PEP), 30 autopsy personnel (50% recommended for PEP), and 21 other persons (4.8% recommended for PEP). Donor contacts represented 188 assessed with 20.2% recommended for PEP, compared with 5.6% of 306 recipient contacts recommended for PEP. Human rabies cases result in substantial use of public health and medical resources, especially when diagnosis is delayed. Although rare, clinicians should consider rabies in cases of encephalitis of unexplained aetiology, particularly for cases that may result in organ donation.

Coat color genotypes and risk and severity of melanoma in gray quarter horses.

BACKGROUND: Both graying and melanoma formation in horses have recently been linked to a duplication in the STX17 gene. This duplication, as well as a mutation in the ASIP gene that increases MC1R pathway signaling, affects melanoma risk and severity in gray horses. OBJECTIVE: To determine if melanoma susceptibility in gray Quarter Horses (QH) is lower than gray horses from other breeds because of decreased MC1R signaling resulting from a high incidence of the MC1R chestnut coat color allele in the QH population. ANIMALS: A total of 335 gray QH with and without dermal melanomas. METHODS: Blood or hair root samples were collected from all horses for DNA extraction and genotyping for STX17, ASIP, and MC1R genotypes. Age, sex, and external melanoma presence and grade were recorded. The effect of age and genotype on melanoma presence and severity was evaluated by candidate gene association. RESULTS: Melanoma prevalence (16%) and grade (0.35) in this QH cohort was lower than that reported in other breeds. Age was significantly associated with melanoma prevalence (P = 5.28 × 10(-11)) and severity (P = 2.2 × 10(-13)). No significant effect of MC1R genotype on melanoma prevalence or severity was identified. An effect of ASIP genotype on melanoma risk was not detected. Low STX17 homozygosity precluded evaluation of the gray allele effect. CONCLUSION AND CLINICAL IMPORTANCE: Melanoma prevalence and severity is lower in this population of gray QH than what is reported in other breeds. This could be because of the infrequent STX17 homozygosity, a mitigating effect of the MC1R mutation on ASIP potentiation of melanoma, other genes in the MC1R signaling pathway, or differences in breed genetic background.

Brain connectivity in body dysmorphic disorder compared with controls: a diffusion tensor imaging study.

BACKGROUND: Several neuroimaging studies have investigated brain grey matter in people with body dysmorphic disorder (BDD), showing possible abnormalities in the limbic system, orbitofrontal cortex, caudate nuclei and temporal lobes. This study takes these findings forward by investigating white matter properties in BDD compared with controls using diffusion tensor imaging. It was hypothesized that the BDD sample would have widespread significantly reduced white matter connectivity as characterized by fractional anisotropy (FA). METHOD: A total of 20 participants with BDD and 20 healthy controls matched on age, gender and handedness underwent diffusion tensor imaging. FA, a measure of water diffusion within a voxel, was compared between groups on a voxel-by-voxel basis across the brain using tract-based spatial statistics within the FSL package. RESULTS: Results showed that, compared with healthy controls, BDD patients demonstrated significantly lower FA (p < 0.05) in most major white matter tracts throughout the brain, including in the superior longitudinal fasciculus, inferior fronto-occipital fasciculus and corpus callosum. Lower FA levels could be accounted for by increased radial diffusivity as characterized by eigenvalues 2 and 3. No area of higher FA was found in BDD. CONCLUSIONS: This study provided the first evidence of compromised white matter integrity within BDD patients. This suggests that there are inefficient connections between different brain areas, which may explain the cognitive and emotion regulation deficits within BDD patients.

In vitro effects of hydrochloric and lactic acids on bioelectric properties of equine gastric squamous mucosa.

REASONS FOR PERFORMING STUDY: Volatile fatty acids, byproducts of carbohydrate fermentation by resident bacteria, have been implicated in causing nonglandular (NG) gastric ulcers. Lactic acid (LA), also produced by stomach bacteria, may cause gastric ulcers when exposed to the equine NG mucosa. OBJECTIVES: To investigate the in vitro effects of LA on equine NG mucosa bioelectric properties, sodium transport and tissue resistance. METHODS: Gastric tissues obtained from 13 mature horses were studied in Ussing chambers. Short-circuit current (Isc) and potential difference (PD) were measured, and electrical resistance (R) and conductance (G) calculated for tissues after addition of HCl and LA (5, 10, 20 and 40 mmol/l) in normal Ringer's solution (NRS). RESULTS: Mucosa exposed to HCl or LA (5, 10 and 20 mmol/l) in NRS (pH 1.5 and to a lesser extent pH 4.0) had a significant decrease in Isc and PD. Mucosa exposed to a high concentration of LA (40 mmol/l) in NRS (LRS) at pH 1.5 showed an increased G, but this increase was not significant. Values returned to baseline after solutions were returned to pH 7.0. Histological changes were consistent with HCl-induced (pH <4.0) acid damage. CONCLUSIONS: HCl induced alteration in bioelectric properties of equine NG mucosa whereas addition of LRS did not, other than those changes seen with HCl alone.

Allergen immunotherapy with heat-killed Listeria monocytogenes alleviates peanut and food-induced anaphylaxis in dogs.

BACKGROUND: Heat-killed Listeria monocytogenes (HKL) potently stimulates interferon (IFN)-gamma production in CD4 T-lymphocytes, and when used as adjuvant for immunotherapy, reduces immunoglobulin (Ig)E production and reverses established allergen-induced airway hyperreactivity (AHR) in a murine model of asthma. We asked if such treatment could decrease established peanut-induced anaphylaxis or cow's milk-induced food allergy in highly food-allergic dogs. METHODS: We therefore studied four 4-year-old atopic colony dogs extremely allergic to peanut (Group I), as well as five 7-year-old dogs very allergic to wheat, milk and other foods (Group II). All dogs experienced marked allergic symptoms, including vomiting and diarrhea on oral challenge with the relevant foods. The dogs were then vaccinated once subcutaneously with peanut or milk and wheat with HKL emulsified in incomplete Freund's adjuvant. RESULTS: Following vaccination of the allergic dogs with HKL and allergen, oral challenges with peanut (Group I) or milk (Group II) elicited only minor or no symptoms. In addition, skin test end-point titrations showed marked reductions for >10 weeks after treatment, and levels of Ara h 1-specific IgE in serum of peanut sensitive dogs, as demonstrated by immunoblotting, were greatly reduced by treatment with HKL plus peanut allergen. CONCLUSIONS: Thus, HKL plus allergen treatment markedly improved established food allergic responses in dogs, suggesting that such an immunotherapy strategy in humans might greatly improve individuals with food allergy and anaphylaxis.

Stable transformation of rice (Oryza sativa L.) via microprojectile bombardment of highly regenerative, green tissues derived from mature seed.

A highly efficient and reproducible transformation system for rice ( Oryza sativa L. cv. Taipei 309) was developed using microprojectile bombardment of highly regenerative, green tissues. These tissues were induced from mature seeds on NB-based medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and high concentrations of cupric sulfate under dim light conditions; germinating shoots and roots were completely removed. Highly regenerative, green tissues were proliferated on the same medium and used as transformation targets. From 431 explants bombarded with transgenes [i.e. a hygromycin phosphotransferase ( hpt) gene plus one of a wheat thioredoxin h ( wtrxh), a barley NADP-thioredoxin reductase ( bntr), a maize Mutator transposable element ( mudrB) or beta-glucuronidase ( uidA; gus) gene], 28 independent transgenic events were obtained after an 8- to 12-week selection period, giving a 6.5% transformation frequency. Of the 28 independent events, 17 (61%) were regenerable. Co-transformation of the second introduced transgene was detected in 81% of the transgenic lines tested. Stable integration and expression of the foreign genes in T(0) plants and T(1) progeny were confirmed by DNA hybridization, western blot analyses and germination tests.

Redox changes accompanying the degradation of seed storage proteins in germinating rice.

The mobilization of storage proteins (glutelins) in germinating rice seeds was accompanied by an ordered sequential combination of proteolysis and reduction of disulfide groups. Mobilization was followed by application of non-reducing/reducing two dimensional-PAGE after monobromobimane labeling of the sulfhydryl groups of the proteins in intact seeds.

A strategy for the identification of proteins targeted by thioredoxin.

Thioredoxins are 12-kDa proteins functional in the regulation of cellular processes throughout the animal, plant, and microbial kingdoms. Growing evidence with seeds suggests that an h-type of thioredoxin, reduced by NADPH via NADP-thioredoxin reductase, reduces disulfide bonds of target proteins and thereby acts as a wakeup call in germination. A better understanding of the role of thioredoxin in seeds as well as other systems could be achieved if more were known about the target proteins. To this end, we have devised a strategy for the comprehensive identification of proteins targeted by thioredoxin. Tissue extracts incubated with reduced thioredoxin are treated with a fluorescent probe (monobromobimane) to label sulfhydryl groups. The newly labeled proteins are isolated by conventional two-dimensional electrophoresis: (i) nonreducing/reducing or (ii) isoelectric focusing/reducing SDS/PAGE. The isolated proteins are identified by amino acid sequencing. Each electrophoresis system offers an advantage: the first method reveals the specificity of thioredoxin in the reduction of intramolecular vs. intermolecular disulfide bonds, whereas the second method improves the separation of the labeled proteins. By application of both methods to peanut seed extracts, we isolated at least 20 thioredoxin targets and identified 5-three allergens (Ara h2, Ara h3, and Ara h6) and two proteins not known to occur in peanut (desiccation-related and seed maturation protein). These findings open the door to the identification of proteins targeted by thioredoxin in a wide range of systems, thereby enhancing our understanding of its function and extending its technological and medical applications.

Evaluation of negation phrases in narrative clinical reports.

OBJECTIVE: Automatically identifying findings or diseases described in clinical textual reports requires determining whether clinical observations are present or absent. We evaluate the use of negation phrases and the frequency of negation in free-text clinical reports. METHODS: A simple negation algorithm was applied to ten types of clinical reports (n=42,160) dictated during July 2000. We counted how often each of 66 negation phrases was used to mark a clinical observation as absent. Physicians read a random sample of 400 sentences, and precision was calculated for the negation phrases. We measured what proportion of clinical observations were marked as absent. RESULTS: The negation algorithm was triggered by sixty negation phrases with just seven of the phrases accounting for 90% of the negations. The negation phrases received an overall precision of 97%, with "not" earning the lowest precision of 63%. Between 39% and 83% of all clinical observations were identified as absent by the negation algorithm, depending on the type of report analyzed. The most frequently used clinical observations were negated the majority of the time. CONCLUSION: Because clinical observations in textual patient records are frequently negated, identifying accurate negation phrases is important to any system processing these reports.

A simple algorithm for identifying negated findings and diseases in discharge summaries.

Narrative reports in medical records contain a wealth of information that may augment structured data for managing patient information and predicting trends in diseases. Pertinent negatives are evident in text but are not usually indexed in structured databases. The objective of the study reported here was to test a simple algorithm for determining whether a finding or disease mentioned within narrative medical reports is present or absent. We developed a simple regular expression algorithm called NegEx that implements several phrases indicating negation, filters out sentences containing phrases that falsely appear to be negation phrases, and limits the scope of the negation phrases. We compared NegEx against a baseline algorithm that has a limited set of negation phrases and a simpler notion of scope. In a test of 1235 findings and diseases in 1000 sentences taken from discharge summaries indexed by physicians, NegEx had a specificity of 94.5% (versus 85.3% for the baseline), a positive predictive value of 84.5% (versus 68.4% for the baseline) while maintaining a reasonable sensitivity of 77.8% (versus 88.3% for the baseline). We conclude that with little implementation effort a simple regular expression algorithm for determining whether a finding or disease is absent can identify a large portion of the pertinent negatives from discharge summaries.

Intelligent aids for parallel experiment planning and macromolecular crystallization.

This paper presents a framework called Parallel Experiment Planning (PEP) that is based on an abstraction of how experiments are performed in the domain of macromolecular crystallization. The goal in this domain is to obtain a good quality crystal of a protein or other macromolecule that can be X-ray diffracted to determine three-dimensional structure. This domain presents problems encountered in real-world situations, such as a parallel and dynamic environment, insufficient resources and expensive tasks. The PEP framework comprises of two types of components: (1) an information management system for keeping track of sets of experiments, resources and costs; and (2) knowledge-based methods for providing intelligent assistance to decision-making. The significance of the developed PEP framework is three-fold--(a) the framework can be used for PEP even without one of its major intelligent aids that simulates experiments, simply by collecting real experimental data; (b) the framework with a simulator can provide intelligent assistance for experiment design by utilizing existing domain theories; and (c) the framework can help provide strategic assessment of different types of parallel experimentation plans that involve different tradeoffs.

Statistical methods for the objective design of screening procedures for macromolecular crystallization.

The crystallization of a new macromolecule is still very much a trial-and-error process. As is well known, it requires the search of a large parameter space of experimental settings to find the relatively few idiosyncratic conditions that lead to diffraction-quality crystals. Crystallographers have developed a variety of screens to help identify initial crystallization conditions, including those based on systematic grids, incomplete factorial and sparse-matrix approaches. These are somewhat subjectively formulated based on accumulated data from past crystallization experiments. Ideally, one would prefer as objective a procedure as possible; however, that requires objective methods that incorporate a broad source of crystallization data. The Biological Macromolecular Crystallization Database (BMCD), a repository of all published crystallization conditions, is an obvious source of this data. This database has been augmented with a hierarchical classification of the macromolecules contained in the BMCD as well as extensive data on the additives used with them. A statistical analysis of the augmented BMCD shows the existence of significant correlations between families of macromolecules and the experimental conditions under which they crystallize. This in turn leads to a Bayesian technique for determining the probability of success of a set of experimental conditions based on the data in the BMCD as well as facts about a macromolecule known prior to crystallization. This has been incorporated into software that enables users to rank experimental conditions for new macromolecules generated by a dense partial factorial design. Finally, an additional advantage of the software described here is that it also facilitates the accumulation of the data required for improving the accuracy of estimation of the probabilities of success - knowledge of the conditions which lead to failure of crystallization.

Insemination of mares with low numbers of either unsexed or sexed spermatozoa.

Two experiments were conducted to determine pregnancy rates in mares inseminated 1) with 5, 25 and 500 x 10(6) progressively motile spermatozoa (pms), or 2) with 25 x 10(6) sex-sorted cells. In Experiment 1, mares were assigned to 1 of 3 treatments: Group 1 (n=20) was inseminated into the uterine body with 500 x 10(6) pms. Group 2 (n=21) and Group 3 (n=20) were inseminated into the tip of the uterine horn ipsilateral to the preovulatory follicle with 25 and 5 x 10(6) pms, respectively. Mares in all 3 groups were inseminated either 40 (n=32) or 34 h (n=29) after GnRH administration. More mares became pregnant when inseminated with 500 x 10(6) (18/20 = 90%) than with 25 x 10(6) pms (12/21 = 57%; P<0.05), but pregnancy rates were similar for mares inseminated with 25 x 10(6) vs 5 x 10(6) pms (7/20 = 35%) (P>0.1). In Experiment 2, mares were assigned to 1 of 2 treatments: Group A (n=11) was inseminated with 25 x 10(6) spermatozoa sorted into X and Y chromosome-bearing populations in a skimmilk extender. Group B (n=10) mares were inseminated similarly except that spermatozoa were sorted into the skimmilk extender + 4% egg yolk. Inseminations were performed 34 h after GnRH administration. Freshly collected semen was incubated in 224 microM Hoechst 33342 at 400 x 10(6) sperm/mL in HBGM-3 for 1 hr at 35 degrees C and then diluted to 100 x 10(6) sperm/mL for sorting. Sperm were sorted by sex using flow cytometer/cell sorters. Spermatozoa were collected at approximately 900 cells/sec into either the extender alone (Group A) or extender + 4% egg yolk (Group B), centrifuged and suspended to 25 x 10 sperm/mL and immediately inseminated. Pregnancy rates were similar (P>0.1) between the sperm treatments (extender alone = 13/10, 30% vs 4% EY + extender = 5/10, 50%). Based on ultrasonography, fetal sex at 60 to 70 d correlated perfectly with the sex of the sperm inseminated, demonstrating that foals of predetermined sex can be obtained following nonsurgical insemination with sexed spermatozoa.

Photoperiodic effects on dairy cattle: a review.

Since the initial report in 1978 of galactopoietic effects of a photoperiod of 16 h of light:8 h of darkness, numerous studies have confirmed long-day stimulation of milk yield. The endocrine factor(s) responsible for increased milk yield, however, has eluded identification. Recent studies suggest that insulin-like growth factor-I (IGF-I) may mediate the galactopoietic response to long day photoperiod. Indeed, long days increase IGF-I in heifers and lactating cows; in the latter case, the response preceded an increase in milk yield. In heifers and cows, the increase in IGF-I is independent of changes in circulating growth hormone. Melatonin feeding to mimic a short-day photoperiod suppressed the increase of IGF-I in heifers induced by long days. However, melatonin feeding had no effect on milk yield in cows. Despite lack of resolution of the endocrine mechanism, dairy producers are interested in how photoperiod management can be integrated with current practices throughout the lactation cycle. There is strong evidence that milk yield responses to long days persist through an entire lactation. Also, long days can be combined with bovine somatotropin (bST) to produce additive increases in milk yield. During the dry period, long days increase the periparturient surge of prolactin. However, relative to long days, short-day treatment during the dry period produces the largest magnitude of response in milk yield during the subsequent lactation. The response to short days during the dry period may be due to a priming effect on the photoperiodic response system. In summary, IGF-I has emerged as a possible mediator of the increase of milk yield in response to long-day photoperiod. Photoperiod can be combined effectively with other management techniques such as bST. Consideration of photoperiod management during the dry period is essential to maximize responses during the subsequent lactation.

Overexpression of thioredoxin h leads to enhanced activity of starch debranching enzyme (pullulanase) in barley grain.

Biochemically active wheat thioredoxin h has been overexpressed in the endosperm of transgenic barley grain. Two DNA constructs containing the wheat thioredoxin h gene (wtrxh) were used for transformation; each contained wtrxh fused to an endosperm-specific B(1)-hordein promoter either with or without a signal peptide sequence for targeting to the protein body. Twenty-two stable, independently transformed regenerable lines were obtained by selecting with the herbicide bialaphos to test for the presence of the bar herbicide resistance gene on a cotransformed plasmid; all were positive for this gene. The presence of wtrxh was confirmed in 20 lines by PCR analysis, and the identity and level of expression of wheat thioredoxin h was assessed by immunoblots. Although levels varied among the different transgenic events, wheat thioredoxin h was consistently highly expressed (up to 30-fold) in the transgenic grain. Transgenic lines transformed with the B(1)-hordein promoter with a signal peptide sequence produced a higher level of wheat thioredoxin h on average than those without a signal sequence. The overexpression of thioredoxin h in the endosperm of germinated grain effected up to a 4-fold increase in the activity of the starch debranching enzyme, pullulanase (limit dextrinase), the enzyme that specifically cleaves alpha-1,6 linkages in starch. These results raise the question of how thioredoxin h enhances the activity of pullulanase because it was found that the inhibitor had become inactive before the enzyme showed appreciable activity.

The use of physician domain knowledge to improve the learning of rule-based models for decision-support.

This paper describes a study testing the hypothesis that the learning of a decision-support model by a computer learning algorithm from clinical data can be improved by the addition of domain knowledge from practicing physicians. The domain of the experiment is community-acquired pneumonia. The overall design of the study compares a computer learning algorithm given clinical data to one given clinical data plus domain knowledge added by physician subjects. This study showed that the performance of the computer-generated models augmented with knowledge added by physician subjects were significantly better than the computer-generated models generated without added knowledge using a two-stage rule induction algorithm in the domain of community-acquired pneumonia. This result was highly significant and shows that the addition of domain knowledge may be beneficial to the learning of clinical decision-support models, especially in domains where data is limited.