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Topic:Laboratory Methods
Laboratory methods in equine research encompass a variety of techniques and procedures used to analyze biological samples from horses to study health, disease, genetics, and physiology. These methods include hematological analyses, biochemical assays, molecular biology techniques, and microbiological cultures. Commonly utilized laboratory techniques involve blood tests for complete blood count (CBC) and serum chemistry, polymerase chain reaction (PCR) for genetic and infectious disease studies, and enzyme-linked immunosorbent assays (ELISA) for detecting specific proteins or antibodies. These methods provide valuable data that contribute to understanding equine health and disease mechanisms. This page compiles peer-reviewed research studies and scholarly articles that explore the application, development, and outcomes of laboratory methods in the context of equine research.
Assessment of the CoaguChek-XS portable prothrombin time point-of-care analyzer for horses. Coagulopathies in horses are common and potentially life-threatening. In equine field medicine, a portable point-of-care (POC) prothrombin time (PT) testing device could be useful to identify early changes in extrinsic clotting. The CoaguChek-XS (Roche Diagnostics) is a small, portable POC PT analyzer used in human medicine. Our preliminary study assessed the suitability of CoaguChek-XS for testing PT in horses and established the PT reference interval (PT RI) in healthy horses using this instrument. Blood samples collected from 102 healthy and ill horses were analyzed with the CoaguChek-XS an...
The Effect of Autologous Protein Solution on the Inflammatory Cascade in Stimulated Equine Chondrocytes. Cartilage injury occurs commonly in equine athletes, often precipitating posttraumatic osteoarthritis (PTOA). Orthobiologics such as autologous conditioned serum (ACS) and autologous protein solution (APS) may be useful in decreasing posttraumatic inflammation, thereby preventing PTOA. The objective of this study was to quantify cytokine concentrations in ACS and APS and evaluate the protective effects of ACS and APS on inflamed chondrocytes cultured . We hypothesized that the combination of platelet-derived growth factors (PDGF) and anti-inflammatory cytokines present in APS would be superior...
Seasonal variation in equine follicular fluid proteome. Proteomic studies of follicular fluid (FF) exist for several species, including the horse; however, the seasonal influence on FF proteome has not been explored in livestock. The application of high-throughput proteomics of FF in horse has the potential to identify seasonal variations of proteins involved in follicle and oocyte growth. Methods: This study (i) profiles the proteomes of equine FF collected from dominant growing follicles during the spring anovulatory season (SAN), and spring (SOV), summer (SUM), and fall (FOV) ovulatory seasons; and (ii) identifies season-dependent regulatory net...
Determination of Salivary Cortisol in Donkey Stallions. Salivary cortisol provides information about free plasma cortisol concentration, and salivary sampling is a noninvasive well-tolerated procedure. The aim of this study was to validate a commercial enzyme immunoassay for the determination of salivary cortisol in donkeys. Saliva samples were collected in four donkey stallions on 13 nonconsecutive days at 8:30 AM to avoid circadian variation. Animals were already accustomed to be handled. Saliva was collected using a swab inserted at the angle of the lips, placed onto the tongue for 1 minute and returned into a polypropylene tube. Tubes were cen...
Modulation of TNF-α, IL-1Ra and IFN-γ in equine whole blood culture by glucocorticoids. Glucocorticoids are important drugs in the treatment of many inflammatory, autoimmune and allergic diseases in humans and animals. We investigated the effects of hydrocortisone and dexamethasone on TNF-α, IL-1Ra and INF-γ release in stimulated whole blood cell culture from healthy horses. Whole blood cell cultures proved to be useful for the characterization of the anti-inflammatory properties of new drugs. Diluted equine whole blood was exposed to lipopolysaccharide (LPS) and PCPwL (a cocktail consisting of phythemagglutinin E, concanavalin A, pokeweed mitogen and lipopolysaccharide) in the...
Evaluation of MALDI-TOF MS and an expanded custom reference spectra database for the identification and differentiation of Taylorella equigenitalis and Taylorella asinigenitalis. Misidentification between Taylorella equigenitalis, the causative agent of contagious equine metritis (CEM), and Taylorella asinigenitalis is observed by the gold standard culture method. The performance of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for Taylorella species identification was evaluated using 85 T. equigenitalis and 28 T. asinigenitalis strains selected on the basis of multilocus sequence typing data. Seven of the T. equigenitalis and 9 of the T. asinigenitalis strains were used to generate in-house reference spectra to expand ...
Equine syndromic surveillance in Colorado using veterinary laboratory testing order data. The Risk Identification Unit (RIU) of the US Dept. of Agriculture's Center for Epidemiology and Animal Health (CEAH) conducts weekly surveillance of national livestock health data and routine coordination with agricultural stakeholders. As part of an initiative to increase the number of species, health issues, and data sources monitored, CEAH epidemiologists are building a surveillance system based on weekly syndromic counts of laboratory test orders in consultation with Colorado State University laboratorians and statistical analysts from the Johns Hopkins University Applied Physics Laborator...
Conformational plasticity of the VEEV macro domain is important for binding of ADP-ribose. Venezuelan equine encephalitis virus (VEEV) is a new world alphavirus which can be involved in several central nervous system disorders such as encephalitis and meningitis. The VEEV genome codes for 4 non-structural proteins (nsP), of which nsP3 contains a Macro domain. Macro domains (MD) can be found as stand-alone proteins or embedded within larger proteins in viruses, bacteria and eukaryotes. Their most common feature is the binding of ADP-ribose (ADPr), while several macro domains act as ribosylation writers, erasers or readers. Alphavirus MD erase ribosylation but their precise contributi...
Concurrent Proteomic Fingerprinting and Molecular Analysis of Cyathostomins. Rapid, cost-effective, efficient, and reliable helminth species identification is of considerable importance to understand host-parasite interactions, clinical disease, and drug resistance. Cyathostomins (Nematoda: Strongylidae) are considered to be the most important equine parasites, yet research on this group is hampered by the large number of 50 morphologically differentiated species, their occurrence in mixed infections with often more than 10 species and the difficulties associated with conventional identification methods. Here, MALDI-TOF MS, previously successfully applied to identify n...
Detection of ESAs in equine urine and blood by SAR-PAGE. Erythropoiesis-stimulating agents (ESAs) have been used in horses for doping purposes to increase the performance of these animals in endurance sports. Currently, enzyme-linked immunosorbent assay (ELISA) and mass spectrometry methods are used to detect ESA abuse in equines. However, the sarcosyl polyacrylamide gel-electrophoresis (SAR-PAGE) technique could also be used, since its application in human doping control is well established and has proven to be more sensitive. In this work, the SAR-PAGE method was used to detect recombinant human erythropoietin (rHuEPO), novel erythropoiesis stimul...
Comparison of two portable clinical analyzers to one stationary analyzer for the determination of blood gas partial pressures and blood electrolyte concentrations in horses. Portable blood gas analyzers are used to facilitate diagnosis and treatment of disorders related to disturbances of acid-base and electrolyte balance in the ambulatory care of equine patients. The aim of this study was to determine whether 2 portable analyzers produce results in agreement with a stationary analyzer. Blood samples from 23 horses hospitalized for various medical reasons were included in this prospective study. Blood gas analysis and electrolyte concentrations measured by the portable analyzers VetStat and epoc were compared to those produced by the cobas b 123 analyzer via conco...
In Vitro Digestibility Measurement of Feedstuffs in Donkeys Using the DaisyII Incubator. Successful studies on in vitro digestibility measurement of feedstuffs with fecal inoculum have been reported for horses, whereas data on donkeys are currently lacking. In this study, we evaluated the use of the Daisy Incubator for in vitro digestibility measurement of feedstuffs using donkey feces as source of microbial inoculum. The method was tested using seven feedstuffs commonly used in donkey diets (alfalfa, bromegrass, ryegrass, and timothy hays; wheat bran and wheat straw; barley grains). Feces were obtained from four female donkeys, and incubations were carried out at 1-week interva...
A luminescence-based assay for evaluating bactericidal antibody to Borrelia burgdorferi in vaccinated horses’ serum. Current serological tests cannot discriminate between bactericidal Borrelia burgdorferi antibodies from others that are merely a response to Borrelia antigenic stimulation. Objective: To develop a sensitive and convenient luminescence-based serum bactericidal assay (L-SBA) to identify serum borreliacidal activity. Methods: Prospective validation study and method comparison. Methods: Serum samples were obtained either from archives of the Animal Health Diagnostic Center at Cornell University (N = 7) or from a vaccination trial (N = 238). Endogenous complement-inactivated serum sample was in...
A new molecular screening tool for the detection of chromosomal abnormalities in donkeys. Chromosomal abnormalities are a major cause of infertility and reproductive problems in equids. Nowadays, their detection is rising due to the use of new diagnostic tools based on molecular markers instead of karyotyping. Reports of this kind of genetic aberrations in domestic donkeys (Equus asinus) are extremely scarce, despite their importance in human activities. In the present study, we analysed the implementation of a short-tandem-repeat (STR)-based molecular method initially developed for horses, as a diagnostic tool to detect chromosomal abnormalities in donkeys. The frequency of five X...
Cryopreservation (-20°C) of equine corneoscleral tissue: Microbiological, histological, and ultrastructural study. To evaluate microbiological, histological, and ultrastructural characteristics of short-term cryopreserved (STC) equine corneoscleral tissue (7 years). Methods: Thirty-four healthy equine globes. Methods: After a decontamination protocol, globes were enucleated and stored at -20°C in broad-spectrum antibiotics. Corneoscleral tissue was evaluated at different storage periods: 1 month-1 year (20 eyes) and 7-9 years (12 eyes). Two eyes were used as controls. Microbiologic study included direct (blood, McConkey, and Sabouraud agars) and enrichment (brain-heart infusion broth) cultures. Cryopr...
Salivary alpha-amylase activity and concentration in horses with acute abdominal disease: Association with outcome. Salivary biomarkers could be useful to objectively evaluate critical illness and prognosis for survival in horses with acute abdominal disease. Objective: To compare salivary alpha-amylase (sAA) activity and concentration in healthy horses and horses with acute abdominal disease, and evaluate the association between sAA activity and concentration with disease severity and outcome. Methods: A prospective cohort. Methods: sAA activity, measured using a colorimetric commercial kit, and concentration, measured using a Time-resolved immunofluorometric assay, in 25 healthy horses and in 33 horses wi...
ECEIM consensus statement on equine metabolic syndrome. Equine metabolic syndrome (EMS) is a widely recognized collection of risk factors for endocrinopathic laminitis. The most important of these risk factors is insulin dysregulation (ID). Clinicians and horse owners must recognize the presence of these risk factors so that they can be targeted and controlled to reduce the risk of laminitis attacks. Diagnosis of EMS is based partly on the horse's history and clinical examination findings, and partly on laboratory testing. Several choices of test exist which examine different facets of ID and other related metabolic disturbances. EMS is controlled ...
Coding sequences of sarcoplasmic reticulum calcium ATPase regulatory peptides and expression of calcium regulatory genes in recurrent exertional rhabdomyolysis. Sarcolipin (SLN), myoregulin (MRLN), and dwarf open reading frame (DWORF) are transmembrane regulators of the sarcoplasmic reticulum calcium transporting ATPase (SERCA) that we hypothesized played a role in recurrent exertional rhabdomyolysis (RER). Objective: Compare coding sequences of SLN, MRLN, DWORF across species and between RER and control horses. Compare expression of muscle Ca2+ regulatory genes between RER and control horses. Methods: Twenty Thoroughbreds (TB), 5 Standardbreds (STD), 6 Quarter Horses (QH) with RER and 39 breed-matched controls. Methods: Sanger sequencing of SERCA reg...
Retraction Note to: Analysis of loxoprofen in tablets, patches, and equine urine as tert-butyldimethylsilyl derivative by gas chromatography-mass spectrometry. The authors have retracted this article [1] because after publication they became aware that the equine urine samples analysed for loxoprofen in this study were in fact equine plasma samples. Therefore the results and conclusions of this article cannot be relied upon. All authors agree to this retraction.
Altered gene expression in embryos and endometrium collected on day 8 of induced aluteal cycles in mares. Aluteal cycles were induced in the mare to evaluate the effects of progesterone deprivation on the gene expression of embryos and endometrium collected eight days after ovulation. We hypothesized that the transcript expression would be altered during induced aluteal (AL) cycles (low progesterone <1 ng/mL) when compared with control cycles during diestrus (high progesterone; > 4 ng/mL) for 1) the embryonic expression of progesterone-mediated transcripts and those related to normal embryo growth and development and 2) the endometrial expression of progesterone-mediated transcripts and those ...
The Identification of Novel Single-Nucleotide Polymorphisms of Equine Beta-Lactoglobulin and Lactotransferrin Genes. Lactoferrin (LTF), also called lactotransferrin, is an iron-binding protein and member of transferrin family, whereas β-LG is an important milk protein and belongs to the ligand-binding protein family of lipocalins and binds retinol. Beta-lactoglobulin (β-LG) is a major whey protein and it is a member of lipocalins family. β-LG is also known as a major allergen reason of cow milk protein allergy. Yet, no information has been reported about β-LG and LTF genes in donkeys. In this study, the genetic variation of β-LG and LTF genes were analyzed via DNA sequencing in 77 donkeys, which were in...
[Comparative analysis of LAMP and Real Time PCR methods to detect pathogens of glanders and meliodosis.]. Results of detection of Burkholderia mallei and Burkholderia pseudomallei DNA strains by LAMP (Loop-mediated Isothermal Amplification) and Real Time PCR are shown. It has been revealed that, in Real Time PCR, primers steadily detected DNA of those microorganism for the sequences of which they were designed. The above mentioned primers did not detect DNA of heterologous strains. During LAMP method no set of primers showed high analytical sensitivity and specificity. Primers did not detected DNA of all the strains under research to target genes of which they were not intended, but they were capa...
Atypical myopathy-associated hypoglycin A toxin remains in sycamore seedlings despite mowing, herbicidal spraying or storage in hay and silage. Several pasture management strategies have been proposed to avoid hypoglycin A (HGA) intoxication in horses, but their efficacy has never been investigated. Objective: To evaluate the effect of mowing and herbicidal spraying on HGA content of sycamore seedlings and the presence of HGA in seeds and seedlings processed within haylage and silage. Methods: Experimental study. Methods: Groups of seedlings were mowed (n = 6), sprayed with a dimethylamine-based (n = 2) or a picolinic acid-based herbicide (n = 1). Seedlings were collected before intervention, and at 48 h, 1 and 2 weeks after. ...
Exosomes isolation and identification from equine mesenchymal stem cells. Mesenchymal stem cells are used for different therapeutic approaches, e.g. for osteoarthritis, lesions of the tendon as well as for bone defects. Current research on the mechanism of stem cells on the repair of damaged tissue suggest an important role of a cell-to-cell communication through secreted extracellular vesicles, mainly represented by exosomes. To enhance the scarce knowledge on the functional role of exosomes we compared as a first step different techniques to isolate and identify exosomes from the supernatant of equine adipose derived mesenchymal stem cells for further characteriza...
Comparative study on the metabolism of the ergot alkaloids ergocristine, ergocryptine, ergotamine, and ergovaline in equine and human S9 fractions and equine liver preparations. 1. Ergopeptine alkaloids like ergovaline and ergotamine are suspected to be associated with fescue toxicosis and ergotism in horses. Information on the metabolism of ergot alkaloids is scarce, especially in horses, but needed for toxicological analysis of these drugs in urine/feces of affected horses. The aim of this study was to investigate the metabolism of ergovaline, ergotamine, ergocristine, and ergocryptine in horses and comparison to humans. 2. Supernatants of alkaloid incubations with equine and human liver S9 fractions were analyzed by reversed-phase liquid-chromatography coupled to h...
Influence of Incubation Time and Incubation Tube on the Cytokine and Growth Factor Concentrations of Autologous Conditioned Serum in Horses. Autologous conditioned serum (ACS) is commonly used for the treatment of human and equine osteoarthritis (OA). The objective of this study was to investigate the influence of incubation time and incubation tube on concentrations of interleukin-1 receptor antagonist (IL-1ra), interleukin-1 beta (IL-1β), and insulin-like growth factor-1 (IGF-1) in ACS of horses with and without OA. Blood samples of 7 control horses and 6 horses with naturally occurring OA were obtained. The concentration of serum IL-1ra, IL-1β, and IGF-1 was determined after 12, 24, and 36 hours of incubation using commercial ...
Expression analysis of miR-221-3p and its target genes in horses. A microRNA (miRNA) is a small non-coding RNA (ncRNA) approximately 20 nucleotides long and it affects gene expression through mRNA cleavage or translational repression. Horses (Equus caballus) have been domesticated and bred to enhance their speed for racing. It has been studied extensively with genetic diversity, origins and evolution. We examined expression patterns of miR-221-3p and its target gene CDKN1C in various horse tissues. We used bioinformatic tools to examine target gene, seed region and evolutionary conservation of miR-221-3p. The expression patterns of miR-221-3p and its target ...
Anti-Horse Podoplanin Monoclonal Antibody PMab-219 is Useful for Detecting Lymphatic Endothelial Cells by Immunohistochemical Analysis. Podoplanin (PDPN) is expressed in lymphatic endothelial cells, where it induces platelet aggregation through C-type lectin-like receptor-2 (CLEC-2). This protein has been characterized for a number of animal species using specific anti-PDPN monoclonal antibodies (mAbs). We recently established the mAb against horse PDPN (horPDPN) named PMab-219. Therefore, in this study, we investigated whether PMab-219 can detect lymphatic endothelial cells in horse tissues. Immunohistochemical analysis demonstrated that PMab-219 strongly stained lymphatic endothelial cells in horse colon tissues, indicating ...
Evaluation of a portable device for assessment of motility in stallion semen. In horse breeding, quality assessment of semen before insemination is often requested. Non-laboratory-based techniques for objective analysis of sperm motility are thus of interest. The aim of this study was evaluating a portable device for semen analysis (Ongo sperm test) and its comparison with computer-assisted semen analysis (CASA). Semen was collected from 10 stallions, diluted to 100, 50 and 25 × 106 sperm/ml and analysed for total (TM) and progressive motility (PM). The final sperm concentration influenced total motility analysed by Ongo (p < 0.05) which was higher at 100 × 10...
An immuno polymerase chain reaction screen for the detection of CJC-1295 and other growth-hormone-releasing hormone analogs in equine plasma. CJC-1295 is a 30 amino acid peptide-based drug that stimulates the release of growth hormone (GH) from the pituitary gland. It is unique among performance-enhancing peptides due to the presence of a reactive maleimidopropionic acid group that covalently links the peptide to free thiols on the surface of plasma proteins. Once conjugated, CJC-1295 remains active in the bloodstream for significantly longer than non-conjugated peptide-based drugs that are rapidly excreted. Conjugation of CJC-1295 to plasma proteins prevents its detection by top-down mass-spectrometry-based peptide screening protoc...
