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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.
Differentiation of respiratory and abortigenic isolates of equine herpesvirus 1 by restriction endonucleases. Viruses classified by immunologic criteria as equine herpesvirus 1 cause respiratory disease and abortion in horses. Restriction endonuclease analyses of the DNA's of viruses from animals with respiratory disease and from aborted fetuses show that the patterns for respiratory viruses, while similar to each other, are entirely different from the patterns for fetal viruses. It is therefore proposed that the DNA restriction endonuclease patterns of fetal and respiratory viruses analyzed in this study be designated as prototypic of equine herpesvirus 1 and 4, respectively.
Primary structure of 3-phosphoglycerate kinase from horse muscle. I. Purification of cyanogen bromide peptides and amino acid sequence of peptide CB5 (104 residues). 3-Phosphoglycerate kinase was isolated from horse muscle and subjected to the action of cyanogen bromide. The resulting peptides were separated using gel filtration combined with either ion exchange chromatography on phosphocellulose in 6 M urea or high voltage paper electrophoresis. The sequence of the largest peptide, CB5, has been determined by a combination of automated and manual Edman degradation carried out on the intact peptide and derivatives obtained by proteolytic digestion. The isolation of two peptides derived from CB5 by cleavage of the bond between Asp109 and Pro110 facilitated ...
Field and laboratory studies of equine influenza viruses isolated in 1979. Experimental ponies developed signs of disease four days after the intranasal instillation of A/England 1/79 equine influenza virus and virus was recovered from the nasopharynx from the second to the ninth day. No significant antigenic difference was found between the virus and the prototype A/Miami 1/63 virus, using post infection ferret and chicken sera and post vaccination pony sera. No antigenic differences were found between four viruses isolated between January and July 1979, although some differences were found in their ability to detect haemagglutination inhibiting antibody in convales...
Effects of cytotoxic drugs on cultured equine cells in vitro. Sixteen cytotoxic drugs used in cancer chemotherapy in man were studied for cytopathic effect on equine fibrosarcoma, melanoma and normal equine lung cells in vitro. Three drugs, vincristine, melphalan and methotrexate, produced cytopathic effect
Antigenic and structural conservation of herpesvirus DNA-binding proteins. Previously, we have shown a common antigen of several herpesviruses (pseudorabies virus, equine abortion virus and bovine mammillitis virus) to be antigenically related to the major DNA-binding proteins of herpes simplex virus types 1 and 2. In this study we have purified the cross-reacting polypeptide from cells infected with pseudorabies virus, equine abortion virus and bovine mammillitis virus and shown the cross-reacting protein to be a major DNA-binding protein for each virus. Tryptic peptide analysis of the cross-reacting DNA-binding proteins of all five viruses has shown structural simi...
Bacteriological and serological studies of haemophilus equigenitalis, agent of contagious equine metritis. Seventeen strains of haemophilus equigenitalis isolated from the cervix, clitoris, and urethra of mares were biochemically characterized with the API 10E and APIZYM test kit systems, conventional biochemical tests, and the porphyrin test. Antisera were prepared in rabbits. All of the strains were positive to the porphyrin test, and the requirement for factor X (hemin) or V (nicotinamide adenine dinucleotide) was not shown. Catalase, oxidase, phosphatase, and phosphoamidase tests were positive with all of the strains. Aminopeptidase (arylamidase) activity has been detected on beta-naphthylamide...
Equine follicle-stimulating hormone. Purification, acid dissociation, and binding to equine testicular tissue. A simple method of purification of equine follicle-stimulating hormone is described by which two forms of the hormone are obtained. The acid dissociation of the most active preparation was studied and a pKa of 5.8 was determined at 37 degrees C. This value is 2 pH units higher than that observed for pregnant mare serum gonadotropin suggesting that the binding areas between subunits are not identical in the two hormones. We also describe an homologous radioreceptor assay of equine follicle-stimulating hormone which is highly specific for this hormone in contrast to the heterologous systems desc...
Mobilization of iron from ferritin by isolated mitochondria. Effects of species compatibility between ferritin and mitochondria and iron content of ferritin. Mitochondria mobilize iron from ferritin by a mechanism that depends on external FMN. With rat liver mitochondria, the rate of mobilization of iron is higher from rat liver ferritin than from horse spleen ferritin. With horse liver mitochondria, the rate of iron mobilization is higher from horse spleen ferritin than from rat liver ferritin. The results are explained by a higher affinity between mitochondria and ferritins of the same species. The mobilization of iron increases with the iron content of the ferritin and then levels off. A maximum is reached with ferritins containing about 1 200 i...
Immunochemical and biological properties of horse parathyroid hormone. No abstract available
Pharmacology of narcotic analgesics in the horse: quantitative detection of morphine in equine blood and urine and logit-Log transformations of this data. Morphine was detected in equine biological fluids by a combination of liquid-liquid extraction and column chromatography, followed by derivatization and gas-liquid chromatographic assay, using electron capture detector. Recovery of morphine from the equine biological samples was poor. However, despite an overall recovery of less than 20%, this method had a detection limit of 0.2 ng/ml. Addition of 5,000 U of bovine liver beta-glucuronidase/ml of urine enabled detection of the drug in urine for up to 144 hours after horses were given 0.1 mg of morphine/kg of body weight. Morphine was found for ...
Isolation and identification of equine lymphocytes and monocytes. Various cell populations of equine mononuclear leukocytes were identified and isolated. Mononuclear leukocytes were concentrated by isopyknic centrifugation, using a solution of Ficoll and Hypaque. Three additional techniques were explored to separate monocytes from lymphocytes, and 3 methods were used to separate lymphocyte types. Cytochemical techniques for the detection of nonspecific esterase readily distinguished equine monocytes from lymphocytes. Peripheral blood lymphocytes were separated into at least 2 populations. One population had surface traits identical to thymocytes [ie, they re...
Plasma progesterone levels in the mare during the oestrous cycle and pregnancy. Plasma progesterone was determined with the aid of a competitive protein-binding assay in mares during the oestrous cycle, early pregnancy (45--60 days) and later pregnancy (2--10 months). Progesterone levels were low during oestrus (less than 1 ng per ml) (3,18 nmol/l) and reached high levels (often in excess of 10 ng per ml) (31.8 nmol/l) within 3--4 days after ovulation. The high luteal levels were maintained for approximately 5--8 days and then declined sharply over a period of approximately 24--48 hours to reach low levels at the subsequent oestrus period. In mares conceiving after servic...
Studies with equine infectious anemia virus: transmission attempts by mosquitoes and survival of virus on vector mouthparts and hypodermic needles, and in mosquito tissue culture. Biological and mechanical transmission trials with Psorophora columbiae (Dyar and Knab) and Aedes sollicitans (Walker) and ponies acutely infected with equine infectious anemia virus (EIAV) were negative. The EIAV antigen was detected by radioimmunoassay in Ae sollicitans immediately after the mosquitoes had fed on an acutely ill pony, but not 14 days after feeding. Psorophora columbiae mosquitoes had detectable EIAV antigen as determined by radioimmunoassay 24 hours after they fed on an acutely ill pony; this antigen was not detected again until 6 days after feeding and was still detected 14 ...
beta-Endorphin: isolation, amino acid sequence and synthesis of the hormone from horse pituitary glands. Beta-endorphin has been isolated from equine pituitaries. Its amino acid sequence is identical to that of ovine, bovine and camel beta-endorphins except for substitution of the threonine residue at position 6 by serine. The equine beta-endorphin has also been synthesized by the solid-phase method. In comparison with the human hormone, equine beta-endorphin was shown to possess 3 times the receptor-binding activity in rat membrane preparations and 1.6 times the analgesic potency in the mouse tail-flick assay.
Diagnosis of eastern equine encephalomyelitis by immunofluorescent staining of brain tissue. Brain tissues were obtained from 5 horses with clinical encephalomyelitis during an epizootic in southwestern Michigan in August-September 1980. These tissues were tested for virus by intracerebral inoculation of suckling mice and by examination of frozen sections and impression smears by the indirect fluorescent antibody (FA) technique. Eastern equine encephalomyelitis virus was isolated and detected by FA technique in brains of 3 horses which died or were euthanatized within approximately 24 hours of onset of the disease but not from 2 horses at 2 and 3 days after onset. The latter 2 animals...
The interaction of equine platelet tropomyosin with skeletal muscle actin. Whereas skeletal muscle tropomyosin binds strongly to muscle F-actin in a buffer containing 30 mM KCl and 1-2 mM free Mg2+, equine platelet tropomyosin only binds stoichiometrically (1 tropomyosin molecule per 6 actin monomers) at higher Mg2+ concentrations (7-8 mM free Mg2+). At low free Mg2+ concentrations (1.5 mM) the binding of the platelet protein is only marginally increased by raising the KCl concentration to an optimal value (0.10-0.20 M). This weaker binding can be attributed to the relatively poor head-to-tail polymerization of platelet tropomyosin and its fewer actin-binding sites. ...
Electron transfer between horse heart and Candida krusei cytochromes c in the free and bound states. Electron transfer between horse heart and Candida krusei cytochromes c in the free and phosvitin-bound states was examined by difference spectrum and stopped-flow methods. The difference spectra in the wavelength range of 540-560 nm demonstrated that electrons are exchangeable between the cytochromes c of the two species. The equilibrium constants of the electron transfer reaction for the free and phosvitin-bound forms, estimated from these difference spectra, were close to unity at 20 degrees C in 20 mM Tris-HCl buffer (pH 7.4). The electron transfer rate for free cytochrome c was (2-3).10(4)...
Amino acid sequence of horse colipase B. The complete sequence of the 96 residues composing horse colipase B has been determined by automated analysis of the intact protein, of two CNBr peptides and two tryptic peptides arising, respectively, from the citraconylated chain and from the unreduced protein. The single histidine of the protein is located at position 29 as in horse colipase A. His86, present in the C-terminal region of the pig cofactor and supposed to play a role in the folding molecule, is not conserved in horse B. Large pieces of the pig and horse B chains were found to be identical or very similar, especially the N-term...
Nucleic acid-protein interactions. Degradation of double-stranded RNA by glycosylated ribonucleases. 1. Extensively glycosylated ribonucleases, like the enzymes from pig and horse pancreas, show a much higher activity on double-stranded RNAs than similarly charged, carbohydrate-free RNAases under stranded assay conditions (relatively high salt concentrations). Glycosylated pig and horse pancreas RNAases also show a larger destabilizing effect on double-stranded poly[d(A-T)] X poly[d(A-T)], than that displayed by bovine RNAase A under these conditions. Both activities show a similar dependence on the ionic strength of the medium. 2. A partial enzymic removal of the heterosaccharide side chains...
Separation and identification of equine leukocyte populations and subpopulations. Various methods of separation and identification of major equine leukocyte populations and subpopulations were used. The purity of T and B lymphocytes separated in Sephadex anti-equine F(ab')2 columns was 87% to 99% and 83% of 97%, respectively. The purity of T lymphocytes separated in nylon-wool columns was 89% to 98%. Preparations of B lymphocytes separated in glass-bead columns were 68% to 79% pure. The presence (or absence) of surface immunoglobulin by immunofluorescence was the most consistent and reliable method for the identification of B or T lymphocytes, respectively. However, the ery...
