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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.
Quantitative analysis of lignocaine and metabolites in equine urine and plasma by liquid chromatography-tandem mass spectrometry. In this paper, a method for the sensitive and reproducible analysis of lignocaine and its four principal metabolites, monoethylxylidide (MEGX), glycylxylidide (GX), 3-hydroxylignocaine (3-HO-LIG), 4-hydroxylignocaine (4-HO-LIG) in equine urine and plasma samples is presented. The method uses liquid chromatography coupled to tandem mass spectrometry operating in electrospray ionisation positive ion mode (+ESI) via multiple reaction monitoring (MRM). Sample preparation involved solid-phase extraction using a mixed-mode phase. The internal standard adopted was lignocaine-d(10). Lignocaine and its...
Evaluation of the Limulus amebocyte lysate and recombinant factor C assays for assessment of airborne endotoxin. As a potent inflammatory agent, endotoxin is a key analyte of interest for studies of lung ailments in domestic environments and occupational settings with organic dust. A relatively unexplored advance in endotoxin exposure assessment is the use of recombinant factor C (rFC) from the Limulus pathway in a fluorometric assay. In this study, we compared airborne endotoxin concentrations in laboratory- and field-collected parallel air samples using the kinetic Limulus amebocyte lysate (LAL) assay and the rFC assay. Air sampling was performed using paired Institute of Occupational Medicine (IOM) sa...
In vitro expression of receptor activator of nuclear factor-kappaB ligand and osteoprotegerin in cultured equine articular cells. To determine concentrations of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) in equine chondrocytes and synoviocytes and to quantify changes in the OPG:RANKL ratio in response to exogenous factors. Methods: Samples of articular cartilage and synovium with grossly normal appearance obtained from metacarpophalangeal and metatarsophalangeal joints of 5 adult (1- to 8-year-old) horses. Methods: Cell cultures of chondrocytes and synoviocytes were incubated with human recombinant interleukin-1beta (hrIL-1beta; 10 ng/mL), lipopolysaccharide (LPS; 10 microg/mL), ...
In vitro antimicrobial activity of gallium maltolate against virulent Rhodococcus equi. The objective of this study was to determine the in vitro antimicrobial activity of gallium maltolate (GaM) against Rhodococcus equi. A total of 98 virulent bacterial isolates from equine clinical cases were examined, of which 19 isolates were known to be resistant to macrolides and rifampin. Isolates were cultured with various concentrations of GaM and minimal inhibitory concentration (MIC) values were determined after 24 and 48 h. Both the MIC(50) and the MIC(90) after 24h of growth were 558 ng/mL (8 μM) and after 48 h of growth were 2230 ng/mL (32 μM). There were no apparent differences b...
Identification of phosphorylation sites of equine beta-casein isoforms. Equine beta-casein is phosphorylated at variable degrees and isoforms carrying 3 to 7 phosphate groups (3P-7P) have been found in milk, but the phosphorylated amino acid residues of each isoform are not yet identified. In the present work, the different phosphorylation variants were first isolated by ion-exchange chromatography and then hydrolysed by trypsin to generate caseinophosphopeptides (CPPs), each containing all the potential phosphorylation sites. The equine CPPs were prepared by metal oxide affinity chromatography, a method based on the affinity of phosphate groups towards titanium d...
A secondary isotope effect study of equine serum butyrylcholinesterase-catalyzed hydrolysis of acetylthiocholine. beta-Secondary deuterium isotope effects have been measured for equine serum butyrylcholinesterase-catalyzed hydrolysis of acetyl-L(3)-thiocholine (L=H or (2)H). The dependencies of initial rates on isotopic substrate concentrations show close adherence to Michaelis-Menten kinetics, and yield the following isotope effects: (D3)k(cat)/K(m)=0.98+/-0.02 and (D3)k(cat)=1.10+/-0.02. The modestly inverse isotope effect on k(cat)/K(m) is consistent with partial rate limitation by a step that converts the sp(2)-hybridized ester carbonyl of the E+A reactant state into a quasi-tetrahedral transition sta...
A comprehensive analysis of germline and expressed immunoglobulin repertoire in the horse. Based on the recently released horse genome, we have characterized the genomic organization of the horse Ig gene loci. The horse IgH locus in genomic scaffold Un0011 contains 40 D(H) segments, 8 J(H) segments and 50 V(H) segments. The Igkappa locus contains only a single C(kappa) gene, 5 J(kappa) segments and a 60 V(kappa) segments, whereas the Iglambda locus contains 7 C(lambda) genes each preceded by a J(lambda) gene segment. A total of 110 V(lambda) segments with the same transcriptional polarity as J(lambda)-C(lambda) were identified upstream of the J(lambda)-C(lambda) cluster. However, 34...
Determination of (13)C/(12)C ratios of urinary excreted boldenone and its main metabolite 5beta-androst-1-en-17beta-ol-3-one. Boldenone (androsta-1,4-dien-17beta-ol-3-one, Bo) is an anabolic steroid known to have been used in cattle breeding or equine sport as a doping agent for many years. Although not clinically approved for human application, Bo or its main metabolite 5beta-androst-1-en-17beta-ol-3-one (BM1) were detected in several doping control samples. For more than 15 years the possibility of endogenous Bo production in human beings has been discussed. This is a challenging issue for doping control laboratories as Bo belongs to the list of prohibited substances of the World Anti-Doping Agency and therefore th...
Evaluation of diagnostic techniques for Anoplocephala perfoliata in horses from Alberta, Canada. Accurate diagnosis of parasites within individual hosts remains a difficult task. Incorrect and/or inaccurate diagnosis restricts the potential for targeted treatment of individuals and limits our understanding of key epidemiological characteristics of potential pathogens of domestic stock. In this study, we compared the specificity and sensitivity of four diagnostic methods for determination of the presence and intensity of the cestode Anoplocephala perfoliata in horses. Over 400 horses from an abattoir in south-central Alberta were evaluated for the presence and number of cestodes. Thirty on...
Isolation and partial sequencing of Equid herpesvirus 5 from a horse in Iceland. Horses are hosts to 2 types of gammaherpesviruses, Equid herpesvirus 2 and 5 (EHV-2 and EHV-5, respectively). Both EHV-2 and EHV-5 are common in horses in Iceland. An Icelandic EHV-5 isolate was recovered by sequential culture in primary fetal horse kidney and rabbit kidney cells. Glycoprotein B, glycoprotein H, and DNA terminase genes of the isolate were fully sequenced, and the DNA polymerase gene was partly sequenced. To date, the glycoprotein B gene of EHV-5 was the only gene that has been reported to be completely sequenced in addition to small parts of the glycoprotein H, DNA polymerase,...
Immunohistochemical study of matrix metalloproteinases-2 and -9, macrophage inflammatory protein-2 and tissue inhibitors of matrix metalloproteinases-1 and -2 in normal, purulonecrotic and fungal infected equine corneas. Determine the effects of matrix metalloproteinases (MMPs)-2, -9, macrophage inflammatory protein-2 (MIP-2), tissue inhibitors of matrix metalloproteinase (TIMP)-1 and -2 by immunohistochemical expression in fungal affected and purulonecrotic corneas. Methods: Paraffin-embedded equine corneal samples; normal (n = 9), fungal affected (FA; n = 26), and purulonecrotic without fungi (PN; n = 41) were evaluated immunohistochemically for MMP-2, -9, MIP-2, TIMP-1 and -2. The number of immunoreactive inflammatory cells was counted and statistics analyzed. Western blot was performed to detect MMP-2, MMP...
Evaluation of a portable clinical analyzer for the determination of blood gas partial pressures, electrolyte concentrations, and hematocrit in venous blood samples collected from cattle, horses, and sheep. To compare results reported for blood gas partial pressures, electrolyte concentrations, and Hct in venous blood samples collected from cattle, horses, and sheep and analyzed by use of a portable clinical analyzer (PCA) and reference analyzer (RA). Methods: Clinically normal animals (24 cattle, 22 horses, and 22 sheep). Methods: pH; Pco(2); Po(2); total carbon dioxide concentration; oxygen saturation; base excess; concentrations of HCO(3)(-), Na(+), K(+), and ionized calcium; Hct; and hemoglobin concentration were determined with a PCA. Results were compared with those obtained for the same bl...
Immunohistochemical analysis of MCT1 and CD147 in equine skeletal muscle fibres. Monocarboxylate transporter 1 (MCT1) and its ancillary protein CD147 facilitate efflux of lactate from the muscle. Expression of MCT1 and CD147 were studied with immunohistochemistry in type I, IIA, IIAB and IIB fibres of equine gluteal muscle. Staining intensity of MCT1 in the cytoplasm as well as in the membranes of fibre types decreased in the order I=IIA>IIAB>IIB and correlated with the oxidative capacity. Capillaries were pronounced in the MCT1 staining. CD147 antibody stained plasma membranes of all fibre types evenly, whereas the staining in the cytoplasm followed that of MCT1. In...
Evaluation of rapid antigen detection kits for diagnosis of equine rotavirus infection. We evaluated antigen detection kits for human rotavirus with regard to their usefulness for diagnosing equine rotavirus infection. Limiting dilution analyses showed that of the seven kits investigated the Dipstick `Eiken' Rota (Dipstick) had the highest sensitivity to two serotypes of equine rotavirus. The Dipstick did not cross-react with several equine intestinal pathogens. An investigation using 249 fecal samples indicated that the sensitivity of the Dipstick was 81.9% and 47.3%, and its specificity was 98.2% and 99.0%, and its concordance rate was 92.8% and 68.3%, compared with values obta...
Identification of cellular proteins interacting with equine infectious anemia virus S2 protein. The macrophage-tropic lentivirus, equine infectious anemia virus (EIAV), encodes the small auxiliary protein S2 from a short open reading frame that overlaps the amino terminus of env EIAV S2 is dispensable for virus replication in cultured cells but is required for disease production. S2 is approximately 7 kDa and has no overall amino acid sequence homology to other cellular or viral proteins. Therefore it is likely that S2 plays a role as an adaptor protein. To further investigate S2 function we performed a yeast-2-hybrid screen to identify cellular proteins that interact with EIAV S2. The s...
Haemotrophic Mycoplasma infection in horses. Haemotrophic mycoplasmas (HM) are parasites on the surface of red blood cells and known to infect a wide range of animals. However, there are no previous evidences of HM infections in horses. In this study HM were detected for the first time in the blood of two horses suffering from poor performance, apathy, weight loss, and anaemia. Using a HM specific PCR assay and subsequent sequencing the infective agents isolated from the blood of said horses were confirmed as closely related to the HM species Mycoplasma haemofelis and 'Candidatus Mycoplasma haemobos'.
[The influence of pH on cholinesterase hydrolysis of alpha-naphthylacetate in the presence of some cationic detergents]. The influence of some cationic detergents on the catalytic activity of the horse blood plasma cholinesterase in reaction of hydrolysis of alpha-naphthylacetate at different pH were investigated. It was shown, that in the absence of detergents in acid pH of the reaction medium the Km value increases, but V remain constant. In the range of pH from 8.5 to 5.0 in the presence of detergents the Km and V values are not practically changed. That is why the activation of cholinesterase hydrolysis of alpha-naphthylacetate in the presence of detergents is considerably higher than that of the neutral pH....
An investigation into the suitability of a commercial real-time PCR assay to screen for Taylorella equigenitalis in routine prebreeding equine genital swabs. Standard bacteriological methods for identifying Taylorella equigenitalis in cervical smears are time consuming. Therefore, a more rapid real-time PCR assay was evaluated for its suitability in screening swabs. Objective: To compare the results of a commercially available real-time PCR assay with routine microbiological culture for the identification of T. equigenitalis, the causative organism of contagious equine metritis, in equine genital swab samples, under 'field trial' conditions. Methods: Routine prebreeding genital swabs (n=2072) collected from Thoroughbred mares and stallions during 2...
Metabolic evaluation of cooled equine spermatozoa. Microscopy has been used in the routine evaluation of sperm metabolism. Nevertheless, it has limited capacity to preview male fertility. As calorimetry may be used to evaluate directly the metabolic activity of a biological system, the aim of this study was to use microcalorimetry as an additive method for sperm metabolism evaluation of cooled equine semen. Two ejaculates of four stallions were collected and motility, viability (eosin 3%) and membrane functional integrity (hyposmotic swelling test) of spermatozoa were evaluated. Sperm samples were processed following different protocols and th...
Identification of differentially expressed genes associated with osteochondrosis in standardbred horses using RNA arbitrarily primed PCR. The aim of this study was to investigate genes for differential expression in cartilage of foals predisposed to osteochondrosis (OC). Tissue was sampled from the cranial part of the distal intermediate ridge of the tibia in the tarso-crural joint. Foals were considered predisposed to OC when parents had OC at the distal intermediate ridge of the tibia. RNA was isolated and subjected to arbitrarily primed PCR (RAP-PCR) followed by fingerprinting to screen for differentially expressed genes. By verification of results from the RAP-PCR fingerprint screening using real-time RT-PCR, we identified t...
Novel markers for tying-up in horses by proteomics analysis of equine muscle biopsies. The aim of the study was to identify new biomarkers for acute tying-up in horses. Skeletal muscle biopsies were taken from 3 horses suffering from acute tying-up and 3 healthy horses. We performed 2D gel electrophoresis and mass spectrometry for identification of proteins that are differentially expressed in tying-up. 2D gel electrophoresis of skeletal muscle sequential extracts yielded more than 350 protein spots on each gel, of which 14 were differentially expressed more than two-fold (p<0.05). In-gel digestion followed by peptide mass fingerprinting enabled identification of three significa...
Comparative in vitro metabolism of the ‘designer’ steroid estra-4,9-diene-3,17-dione between the equine, canine and human: identification of target metabolites for use in sports doping control. Effective detection of the abuse of androgenic-anabolic steroids in human and animal sports often requires knowledge of the drug's metabolism in order to target appropriate urinary metabolites. 'Designer' steroids are problematic since it is difficult to obtain ethical approval for in vivo metabolism studies due to a lack of a toxicological profile. In this study, the in vitro metabolism of estra-4,9-diene-3,17-dione is reported for the first time. This is also the first study comparing the metabolism of a designer steroid in the three major species subject to sport's doping control; namely th...
Evaluation of a new enzyme-linked immunosorbent assay to detect keratan sulfate in equine serum. This study aimed to evaluate a system that identifies cartilage turn over and/or degradation through measurement of a new keratan sulfate (KS) epitope concentration in equine sera. Blood samples were collected from 30 horses, 1 (n = 15) and 2 year-olds (n = 15). Serum samples were analyzed for an epitope of keratan sulfate by 1/20/5D4 (KS5D4) and new epitopes of keratan sulfate using high sensitive keratan sulfate (HSKS), measured by two respective enzyme-linked immunosorbant assays (ELISAs). There was no correlation in serum concentration of KS evaluated using 5D4 and HSKS. Age had no signifi...
Enantioselective CE analysis of hepatic ketamine metabolism in different species in vitro. Ketamine, an injectable anesthetic and analgesic consisting of a racemic mixture of S-and R-ketamine, is routinely used in veterinary and human medicine. Nevertheless, metabolism and pharmacokinetics of ketamine have not been characterized sufficiently in most animal species. An enantioselective CE assay for ketamine and its metabolites in microsomal preparations is described. Racemic ketamine was incubated with pooled microsomes from humans, horses and dogs over a 3 h time interval with frequent sample collection. CE data revealed that ketamine is metabolized enantioselectively to norketamine...
Identification and differentiation of Taylorella equigenitalis and Taylorella asinigenitalis by lipopolysaccharide O-antigen serology using monoclonal antibodies. Lipopolysaccharides (LPSs) from Taylorella equigenitalis, the causative agent of contagious equine metritis, and T. asinigenitalis were compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Lipopolysaccharide profiles of 11 T. equigenitalis strains were similar, but different from the profiles of 3 T. asinigenitalis strains, and the profiles of 2 T. asinigenitalis strains were similar to each other. The serological specificities of the LPSs from these 14 strains were examined by immunoblotting and enzyme-linked immunosorbent assay with monoclonal antibodies (MAbs) ...
Direct injection horse-urine analysis for the quantification and confirmation of threshold substances for doping control. IV. Determination of 3-methoxytyramine by hydrophilic interaction liquid chromatography/quadrupole time-of-flight mass spectrometry. Levodopa and dopamine have been abused as performance-altering substances in horse racing. Urinary 3-methoxytyramine is used as an indicator of dopaminergic manipulation resulting from dopamine or levodopa administration and is prohibited with a urinary threshold of 4 microg mL(-1) (free and conjugated). A simple liquid chromatographic (LC)/mass spectrometric (MS) (LCMS) method was developed and validated for the quantification and identification of 3-methoxytyramine in equine urine. Sample preparation involved enzymatic hydrolysis and protein precipitation. Hydrophilic interaction liquid chro...
Immunochromatographic lateral flow test for detection of antibodies to Equine infectious anemia virus. The purpose of this study was to develop and evaluate a simple immunochromatographic lateral flow (ICLF) test for specific detection of Equine infectious anemia virus (EIAV) antibodies in equine sera. Viral recombinant p26 capsid protein (rp26) was used as the capture protein in the test line and as the detector reagent conjugated to colloidal gold. The performance of rp26-ICLF was evaluated, and the results obtained were compared with a commercially available agar gel immunodiffusion (AGID) test used as a standard of comparison according to international guidelines. The values obtained for co...
Fine-mapping and mutation analysis of TRPM1: a candidate gene for leopard complex (LP) spotting and congenital stationary night blindness in horses. Leopard Complex spotting occurs in several breeds of horses and is caused by an incompletely dominant allele (LP). Homozygosity for LP is also associated with congenital stationary night blindness (CSNB) in Appaloosa horses. Previously, LP was mapped to a 6 cm region on ECA1 containing the candidate gene TRPM1 (Transient Receptor Potential Cation Channel, Subfamily M, Member 1) and decreased expression of this gene, measured by qRT-PCR, was identified as the likely cause of both spotting and ocular phenotypes. This study describes investigations for a mutation causing or associated with the Le...
