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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.
High-performance liquid affinity chromatography on silica-bound alcohol dehydrogenase. Horse liver alcohol dehydrogenase was immobilized on glycerylpropyl-silica (10 micron, 1000-A pores) activated with 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride). The coupling and activity yield was almost 100%. The coenzyme-binding sites were equivalent and virtually unaffected by the immobilization process, as judged from Scatchard plots and active-site titrations. The silica-bound enzyme, packed in steel columns, was integrated with HPLC equipment and then successfully used for chromatography of adenine nucleosides, adenine nucleotides, and triazine dyes. Dissociation constants w...
Enzymatic trimethylation of lysine-72 in cytochrome c. The present observations are the continuation of our earlier study on the physicochemical mechanism of protein-lysine methylation. In this paper the electrophoretic behaviour (pI values) of two chemically modified horse heart cytochromes c at lysine-72 with trifluoromethylphenylcarbamoyl (neutral group) or carboxydinitrophenyl (acidic group) is compared with the enzymatically methylated cytochrome c. The results indicate that although both chemically modified cytochromes c have lower pI values than the unmodified cytochrome c, the enzymatic methylation appears to be much more efficient in lowe...
Measurement of superoxide dismutase, diamine oxidase and caeruloplasmin oxidase in the blood of thoroughbreds. Methods were developed for the measurement of superoxide dismutase (SOD), diamine oxidase (DAO) and caeruloplasmin oxidase in the blood of thoroughbred horses. These enzymes were measured in 178 normal thoroughbreds stabled throughout the United Kingdom. The relationships between the activities of SOD, DAO and caeruloplasmin oxidase and the blood concentrations of their associated trace metals (copper, zinc and manganese) were studied in 52 of the thoroughbreds. Trace metals were measured by electrothermal atomic absorption spectrophotometry. No relationships were found between the activities ...
Sodium and potassium ion-dependent change in oligomerization of Na,K-ATPase in C12E8 detected by low-angle laser light scattering technique in combination with high performance porous silica-gel chromatography. Approximate molecular weights and the subunit structures of Na,K-ATPase from horse kidney were estimated by means of the combination of porous silica gel chromatography, laser light scattering (LS) and refractive index (RI) measurements in C12E8. When the enzymes were eluted with NaCl- or KCl-containing solution, 3 or 4 protein peaks, respectively were detected except that of low molecular weight range. These peaks were tentatively named Na-1, Na-2, Na-2', Na-3 (NaCl-containing eluents), K-1, K-2, K-3 (KCl-containing eluents), respectively. Na,K-ATPase and K-p-nitrophenylphosphatase activities...
The in vitro effects of EDTA-tris, EDTA-tris-lysozyme, and antimicrobial agents on equine genital isolants of Pseudomonas aeruginosa. Five isolants of Pseudomonas aeruginosa collected from clinical cases of equine genital infection and one standard strain of P. aeruginosa were exposed to various concentrations of ethylene-diaminetetraacetic acid (EDTA) and tris (hydroxymethyl) aminomethane (tris buffer pH 8) and EDTA-tris lysozyme. Colony forming units of the isolants and minimal inhibitory concentrations for 11 antimicrobial agents were determined with each isolant before and after exposure to the EDTA solutions. Decreased cellular viability was found with all six isolants after exposure to the EDTA-tris solutions. Reversal...
Unfolding pathway of myoglobin. Evidence for a multistate process. The free energy of unfolding of horse myoglobin has been calculated from the denaturation pattern induced by guanidine hydrochloride as well as by acid. The delta GH2O, i.e., the value in the absence of denaturant obtained by using the two-state transition model, was found to be 25% lower than that determined from the acid denaturation pattern, i.e., 12.0 kcal/mol, although the extent of protein denaturation produced by acid was much lower. The amount of helical structure surviving the acid-induced conformational change was estimated to be 50% of that present in the native protein, and it coul...
Interspecies activation of lecithin-cholesterol acyltransferase by apolipoprotein A-I isolated from the plasma of humans, horses, sheep, goats and rabbits. The abilities of apolipoprotein A-I species isolated from humans, horses, sheep, goats and rabbits to activate purified human lecithin-cholesterol acyltransferase and the enzyme from homologous plasmas and plasma of other mammalian species were compared. Each purified apolipoprotein A-I species was individually incorporated into phosphatidylcholine/cholesterol vesicles by the cholate dialysis method to form proteoliposome common substrates (apolipoprotein A-I/phosphatidylcholine/cholesterol molar ratio of 1:250:12.5) for the enzyme activity assay. All apolipoprotein A-I species tested had the ...
Concentration of nucleotides and deoxynucleotides in peripheral and phytohemagglutinin-stimulated mammalian lymphocytes. Effects of adenosine and deoxyadenosine. Concentrations of purine and pyrimidine ribonucleotides were measured with HPLC in lymphocytes of man, horse, pig and sheep and in rat thymocytes. The ATP concentration was highest in lymphocytes of all species and about 850 pmol/10(6) cells in human and equine lymphocytes, higher in porcine and lower in ovine lymphocytes and rat thymocytes. The GTP concentration was comparable in human, equine and porcine lymphocytes, but lower in ovine lymphocytes. ATP concentration was also measured in lymphocytes of man, horse and pig with a luciferin-luciferase assay. During culturing with or without phyt...
Serodiagnosis of western equine encephalitis virus infections: relationships of antibody titer and test to observed onset of clinical illness. Sera from horses and human beings with clinically diagnosed western equine encephalitis (WEE) virus infections were tested for hemagglutination-inhibition (HI), complement-fixation (CF), and neutralizing (N) antibody to WEE virus. These tests confirmed infection in 43.8% (HI), 56.3% (CF), and 80.4% (N) of horses and 54.5% (HI), 59.1% (CF), and 77.3% (N) of human beings. Use of the N test as an adjunct to the HI and CF tests increased the likelihood of serologic confirmation to 91.7%. In both horses and human beings, N antibody increased steeply at the end of the 1st week after onset. The resul...
Simplified technique for histochemical determination of three fiber types in equine skeletal muscle. For determination of 3 muscle fiber types in equine skeletal muscle, a comparison of 2 preincubation buffers, each followed by myosin adenosine triphosphatase staining, was made. Serial sections of the muscle samples (n = 75) were preincubated in an acid buffer (pH 4.6) or a formaldehyde-glycine buffer (pH 7.25) and then were stained for myosin adenosine triphosphatase. Differentiation of muscle fibers into type I, IIA, and IIB was identical with both techniques; however, in the samples prepared at pH 4.6, type I fibers were black; type IIA, light gray; and type IIB, dark gray. In the samples ...
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...
