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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.
Serum albumin values from healthy cattle, sheep and horses determined by the immediate bromocresol green reaction and by agarose gel electrophoresis. Serum albumin concentrations were measured by the immediate reacting bromocresol green (BCG) method and by agarose gel electrophoresis in healthy cattle, sheep and horses. No statistically significant differences were found between the values obtained by the two methods. The immediate reacting BCG method is quicker and cheaper when used under the conditions described.
Detection of rotavirus in horses with and without diarrhea by electron microscopy and Rotazyme test. A total of 142 equine fecal samples (93 field fecal and 49 experimental fecal specimens) were examined for rotavirus using direct electron microscopy (EM) and the Rotazyme test. Eighty-six stool specimens were diarrhea samples. The Rotazyme test sensitivity and accuracy as compared to EM was determined by the visual (color reaction) and spectrophotometric methods. The overall agreement was 94.8% and 92.3% between EM and Rotazyme visual and spectrophotometric methods, respectively when suspect reactions (1 + color reaction or net absorbance between 0.05 and 0.1) were not included. The Rotazyme ...
A collaborative assay of mycoplasma reference antisera. A total of 29 Mycoplasma and Acholeplasma antiserum reagents produced in mules and horses by the Baltimore Biological Laboratory and by Huntingdon Research Centre, under the auspices of National Institutes of Health, Bethesda, USA, were tested for potency and specificity, by a great variety of serological techniques, at the FAO/WHO Collaborating centre for Animal Mycoplasmas, University of Aarhus, Denmark. Subsequently, the antisera were subjected to a collaborative assay in which 20 workers from 15 different laboratories participated under the auspices of the International Research Programme ...
Radioimmunological measurement of beta-endorphin in equine plasma. Radioimmunoassay procedures were developed and validated for the quantification of beta-endorphin (beta-EP)-like immunoreactivity in equine plasma. beta-EP could be quantitatively extracted from plasma with silicic acid powder and subsequently assayed, however, valid estimates of this hormone could also be obtained on unextracted plasma. Although beta-lipotropin (beta-LPH) cross-reacted in the assay, it was not necessary to correct for beta-LPH activity when assaying unextracted plasma because chromatographic analyses showed that 92% of the immunoreactivity in plasma extracts was similar in mo...
Further purification and characterisation of horse IgE. Horse IgE was isolated from a serum pool collected from foals naturally infected with endoparasites. The serum was precipitated with ammonium sulfate, delipidated with dextran sulfate and further purified by gel filtration, anionic exchange, immunosorption or preparative polyacrylamide gelelectrophoresis. By these methods IgE could be isolated at a purity of 81%. The sera from rabbits immunized with the purified horse serum fractions were tested using reversed passive cutaneous anaphylaxis and an enzyme linked immunosorbent assay (ELISA). By the ELISA method cross reaction of rabbit anti horse...
Studies related to the metabolism of anabolic steroids in the horse: the metabolism of 1-dehydrotestosterone and the use of fast atom bombardment mass spectrometry in the identification of steroid conjugates. The metabolism and urinary excretion of 1,2(n)-3H-1-dehydrotestosterone were studied in cross-bred gelded horses. Approximately 40% of the dose was excreted in 24 h. The steroid metabolites were extracted by Amberlite XAD-2 resin and fractionated into glucuronides and sulphoconjugates. Unchanged 1-dehydrotestosterone was the only component identified by gas chromatography mass spectrometry after solvolysis of the sulphoconjugates. Positive and negative ion fast atom bombardment mass spectra were obtained on the purified 1-dehydrotestosterone sulphoconjugate isolated from horse urine and on the...
Allergen-specific ELISA for horse IgE. An enzyme-linked immuno sorbent assay (ELISA) for measuring horse IgE specific to ovalbumin, bencylpenicilloic acid and odinitrocarboxyphenol is described. We used a sandwich type of ELISA by which horse serum was incubated in antigen-coated tubes containing one additional polystyrene ball, followed by rabbit anti horse IgE serum. The tubes were then incubated with biotinylated goat anti rabbit globulin followed by avidin coupled to phosphatase. Endpoint titrations were compared. The ELISA is highly reproducible due to the pretreatment of the polystyrene with glutaraldehyde. The increased anti...
[Study of conformational changes in alcohol dehydrogenase during its interaction with silochrome adsorbent by the EPR spectroscopy method]. The possible use of EPR spectroscopy (spin labelling) for the study of horse liver alcohol dehydrogenase with a silochrome adsorbent is discussed. The rotatory diffusion of nitroxyl labels chemically linked to the enzyme was studied with reference to the time of the enzyme incubation with the adsorbent and the degree of its accumulation on the adsorbent surface. The mobility of nitroxyl radicals attached to the protein globules was shown to increase with time. It was concluded that the conformation of the enzyme molecules changes during their interaction with the adsorbent.
Chemiluminescence response of equine alveolar macrophages during stimulation with latex beads, or IgG-opsonized sheep red blood cells. Isolated equine alveolar macrophages were shown to generate a luminol-dependent light response when challenged with a phagocytic stimulus. The chemiluminescent response was not detected with luminol prepared at 1.0 x 10(-5) or 1.0 x 10(-4) molar concentrations, but was readily quantitated when used at a 1.0 x 10(-3) molar concentration. Challenge of the alveolar macrophages with latex particles or with equine IgG-coated sheep red blood cells elicited the luminol-dependent light response, whereas unchallenged equine alveolar macrophages or those challenged with unopsonized erythrocytes failed t...
[The value of blood progesterone determination about 18 days post ovulation for pregnancy testing in mares]. The reliability of determination of the plasma progesterone level within approximately eighteen days after ovulation in the pregnancy diagnosis of mares is examined in the present study. Studies were done in seventy-five mares, a number of which were served or inseminated during several cycles so that a total number of eighty-seven blood samples were obtained. On the analogy of other authors, the progesterone level above which mares were believed to be pregnant and below which they were assumed to be non-pregnant, was set at 2 ng/ml. The twenty-five mares in which the level was below 2 ng/ml. ...
Localization of the second calcium ion binding site in porcine and equine phospholipase A2. At alkaline pH porcine pancreatic phospholipase A2 is known to bind two Ca2+ ions per protein molecule. One Ca2+ ion is strongly bound to the active site and is essential for enzyme activity. A second Ca2+ ion binds more weakly to the protein and improves the affinity of the enzyme for lipid-water interfaces severalfold at high pH values. A group having a pK around 6 controls enzyme binding to lipid-water interfaces in the absence of Ca2+. By use of proton titration techniques this group is now identified to be a carboxylate having an abnormally high pK. Its pK shifts to a value around 4.5 in ...
The fluorescent antibody technique for diagnosis of equine histoplasmosis (epizootic lymphangitis). Summary
The fluorescent antibody technique (FA) was evaluated as a diagnostic aid for the diagnosis of equine histoplasmosis, using sera from ten clinical cases of epizootic lymphangitis. The globulin fraction was separated, quantitated and then labelled with fluorescein isothiocynate. Of several antigen fixatives, heat-fixation was the best. Conjugated sera diluted 1:5 yielded the highest fluorescence intensity with heat-fixed mycelial antigen preparations. Acetone and P-formaldehyde treated preparations gave a less efficient intensity. Glutaraldehyde was the least effective fixative.
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An in vitro study on joint fitting and cartilage thickness in the radiocarpal joint of foals. The change in the degree of fitting of the opposing articular cartilage surfaces of the radiocarpal joint of foals was studied in vitro using a pneumatic loading device. The thickness of the articular cartilage of the distal radius was examined using radiographic and histological techniques. The thickest cartilage corresponded with the approximate centre of the contact area of the opposing articular surface. The variations in articular cartilage contact area and thickness are discussed.
Isolation of horse mononuclear cells, especially of monocytes, on Isopaque-Ficoll neutral density gradient. Horse mononuclear cells were separated from whole blood using neutral density gradient centrifugation on Isopaque-Ficoll. The resulting cell suspension was comparable in composition with similarly prepared human and bovine mononuclear cell preparations. The relative concentration of monocytes was increased by the use of a gradient with density lower than that originally proposed by Böyum (Böyum, A. 1968. Scand. J. Clin. Lab. Investig. 21 supple. 97:77-89). Contamination by neutrophils was limited either by using a gradient medium of lower density or by replacing Isopaque-Ficoll by Percoll-0....
Adaptation of human diploid fibroblasts in vitro to serum from different sources. The growth of two human diploid skin fibroblast cell lines, originally grown in medium supplemented with foetal bovine serum and later adapted to medium supplemented with newborn bovine, bovine calf or horse serum, has been studied. Prolonged generation times increased cell volumes and decreased plating efficiencies were observed in cultures grown in newborn bovine, bovine calf or horse serum. In general, the deleterious effects were most severe as a result of growth in bovine calf or horse serum. In the light of the present findings, we believe investigators should exert great caution in swit...
Selective crystallization of horse isoferritins. Various precipitating agents were examined in order to crystallize horse heart and spleen ferritins. Cadmium sulfate induced the crystallization of the spleen ferritin, while 2-methyl-2,4-pentanediol and poly(ethylene glycol) only induced that of the heart ferritin. Isoelectric focusing analysis showed that the crystals grown from cadmium sulfate contained only the more acidic isoferritins, and those grown from methyl pentanediol only the less acidic isoferritins. Heart ferritin crystallizes in a cubic space group, as previously reported for spleen ferritin crystals grown from cadmium sulfate....
Laboratory investigations on equine influenza during the outbreak in Poland in 1980. Summary
The outbreak of equine influenza in Poland in 1980 was caused by an equine influenza virus antigenically related to the strain A/equine/Miami/63/Heq2, Neq2. This was confirmed by isolation of six strains of influenza virus from horses suffering from the acute form of the disease. About 45 % and 69 % of positive results were obtained in the HI test performed with sera taken from convalescent and affected animals, respectively. A relatively high level of antibodies against newly isolated equine influenza virus strains was found in 50 % of serum samples taken from the grooms. A relativ...
Alterations in the equine herpesvirus 1 genome after in vitro and in vivo virus passage. The effect of in vitro and in vivo serial virus passage on the genetic stability of equine herpesvirus 1 (EHV-1) was investigated by restriction endonuclease analysis of the viral DNA. DNAs of EHV-1 isolates at different passage levels in cultured cells or in Syrian hamsters were compared by electrophoresis of the DNA cleavage fragments produced by restriction endonuclease digestion. No changes were observed in the restriction profile of the DNAs of EHV-1 strains after 100 sequential passages in cultured equine cells. However, serial passage of the virus in hamsters or in cells of non-equine o...
Use of procainamide gels in the purification of human and horse serum cholinesterases. Two large-scale methods based primarily on the use of procainamide-Sepharose gels were developed for the purification of horse and human serum non-specific cholinesterases. With method I, the procainamide-Sepharose 4B gel was used in the first step to handle large volumes of serum. With method II, the procainamide-Sepharose 4B gel was used in the final step to obtain pure enzyme. Although both methods gave electrophoretically pure cholinesterase preparations in good yields, they were significantly more efficient at purifying the horse enzyme than the human enzyme. To study this problem, the re...
Examination of the origin of increased equine serum alkaline phosphatase concentrations. Serum alkaline phosphatase activity was found to be increased in 32.6% of equine samples analyzed at the Ontario Veterinary College over an 18 month period. An attempt was made using sensitivity to L-phenylalanine and heat to identify the origin of increased serum alkaline phosphatase isoenzymes present in 44 clinical cases. No difference in sensitivity to either procedure was observed for serum alkaline phosphatase from groups of foals and horses representing different clinical problems. Alkaline phosphatase of osseous tissue origin appeared to be the major source of activity for each group o...
Identification and measurement of testosterone in plasma and follicular fluid of the mare, using gas chromatography-mass spectrometry associated with isotope dilution. Testosterone has been identified by mass spectrometry in blood and follicular fluid aspirated from mature Graafian follicles of mares. Quantitative measurements made by gas chromatography-mass spectrometry have validated the determination of plasma testosterone made by radioimmunoassay. However, because of high levels of epitestosterone (17 alpha-hydroxyandrost-4-en-3-one) in the follicular fluid, radioimmunoassay overestimates the true concentrations of testosterone. The occurrence of testosterone in mare follicular fluid at a concentration which is two orders of magnitude higher than that in...
A comparative study of the effect of triazine herbicides on alcohol dehydrogenases isolated from various sources. The studied herbicides (terbutylazine, simazine) inhibit the activity of plant, animal, and yeast alcohol dehydrogenases. The inhibition constant Ki for alcohol dehydrogenase (ADH) isolated from peas and bakers' yeast equals approximately 10(-4) M, and that for ADH isolated from horse liver is of the order of 10(-5) M. The character of inhibition for all the herbicides studied for the reaction catalyzed by pea, liver, and yeast ADH is always noncompetitive toward ethanol and competitive with respect to NAD. The inhibition constants for the enzyme isolated from peas are pH independent. The inte...
Dansylarginine N-(3-ethyl-1.5-pentanediyl)amide. A potent and selective fluorescent inhibitor of butyrylcholinesterase. Interactions between dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA) and the cholinesterases were examined by the techniques of enzyme kinetics and fluorescence spectroscopy. When tested with partially purified enzyme preparations, DAPA was a potent inhibitor of butyrylcholinesterase (IC50 = 2 x 10(-7) M) but not of acetylcholinesterase (IC50 = 4 x 10(-4) M). For a detailed study of the effects of DAPA on butyrylcholinesterase (BuChE), the enzyme was purified to homogeneity from horse serum, with the aid of affinity chromatography on N-methyl acridinium. The kinetics of the inhibition o...
Difference in sizes of human compared to murine alpha-subunits of the glycoprotein hormones arises by four-codon gene deletion or insertion. The sizes of the human and subhuman alpha-subunits of the glycoprotein hormones differ by four amino acids (hCG alpha, 92 amino acids; murine, equine, bovine, and ovine alpha, 96 amino acids). The shortening of the human alpha-subunit has been attributed to posttranslational proteolysis. We have recently determined the nucleotide sequences of the mRNAs encoding the precursors of the alpha-subunit of mouse TSH and rat gonadotropins using recombinant DNA techniques. In this report, we have compared these nucleotide sequences and their deduced amino acid sequences with those of the pre- alpha-sub...
Further study of the chemical structure of the equine erythrocyte hematoside containing O-acetyl ester. The chemical structure of an equine hematoside, which contained an ester group and comprised 72% of the total erythrocyte gangliosides, was determined by means of nondestructive and destructive procedures. A 400-MHz nuclear magnetic resonance spectrum of the ganglioside in perdeuterodimethyl sulfoxide demonstrated three protons due to a methyl group of an acetyl moiety, as well as amide and anomeric protons which were compatible with those of the ordinary hematoside. The spin decoupling difference spectroscopy of the ganglioside revealed the presence of the following structures. [formula: see ...
Investigations into the biology of three ‘phycomycotic’ agents pathogenic for horses in Australia. Although 'phycomycosis' is a common disease of horses in northern Australia little is known about the causative fungi. In this paper the laboratory methods for diagnosis are described. These revealed 38 cases caused by Pythium sp. (Hyphomyces destruens), 6 cases caused by Basidiobolus haptosporus and 2 caused by Conidiobolus coronatus. Laboratory studies on the chemotatic behaviour of zoospores of Pythium sp. showed that they were strongly attracted to both animal hairs and plant tissue. Because of this behaviour a simple baiting method using human hair was used to trap the fungus from water s...
