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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.
Coagulation studies f plasmas from healthy domesticated animals and persons. Optimal reaction conditions for the activated partial thromboplastin time (APTT), 1-stage prothrombin time (PT), and Russell's viper venom time were studied for pooled plasmas of horses, cattle, sheep, goats, swine, dogs, cats, and persons. Changes in CaCl2 and NaCl concentrations had significant effects on the APTT and PT reactions. The APTT was more sensitive than the PT to changes in CaCl2 concentration. The CaCl2 concentration recommended by the manufacturer for the APTT was suboptimal for some of plasmas of domesticated animals in ths study. Infusorial earth (Celite, activator) concentrat...
Gas/liquid chromatographic analysis of pemoline in biological fluids using electron capture detection. An analytical gas/liquid chromatographic (GLC) protocol is described for the quantitation of pemoline in biological fluids of the horse. Plasma samples containing known quantities of pemoline and its analog as an internal standard (IS) were deproteinized with 5-sulfosalicylic acid, heated at 80 degree C, and centrifuged. 5-Phenyl-2,4-oxazolidinedione, the hydrolytic product of pemoline in acid medium, was extracted with dichloromethane (DCM). The organic layer was in turn re-extracted with 1% NaHCO3. The aqueous layer was acidified with HCI, and re-extracted with DCM, which was evaporated to d...
Diagnostic methods in infectious respiratory disease. For laboratory diagnosis of respiratory disease it is of overwhelming importance that the specimens taken are adequate, taken from the correct site and at the correct time. The lower regions of the respiratory tract are particularly difficult to sample but are more likely to yield the causative agent of a pneumonia. Infections involving the upper respiratory tract are much easier to sample and appropriate aspiration apparatus can be used. Consideration must be given to the timing of sample collection in relation to the life cycle of the causative micro-organism. Sampling of several animals is ...
Serologic and molecular comparisons of several equine herpesvirus type 1 strains. The molecular and serologic relatedness of 2 recent respiratory tract isolates of equine herpesvirus type 1, designated T1 and T2, were compared with the Army 183, Kentucky-A hamster-adapted (KyA-ha), and L-M cell-adapted (KyA-LM) strains. Electrophoresis in polyacrylamide gels revealed differences in virion structural proteins among 4 purified strains. Seven envelope glycoproteins (molecular weight of 93,000, 65,000, 62,000, 60,000, 36,000, 20,000, and 18,000) corresponding to virion proteins 13, 16, 17, 18, 23, 25, and 26a, respectively, found in both the Army 183 and KyA-ha strains had slig...
Analysis of the genome of equine herpesvirus type 1: arrangement of cleavage sites for restriction endonucleases EcoRI, BglII and BamHI. The genome of an Australian isolate of equine herpesvirus type 1 (equine abortion virus) has been analysed using the restriction endonucleases EcoRI, BglII and BamHI, and a physical map constructed. Terminal fragments were identified by exonuclease treatments, and linkage of fragments was deduced by a combination of single- and double-digest experiments and cross-blot hybridizations. The genome has a mol. wt. of 100 x 10(6) and is comprised of a short unique region bounded by repetitive sequences, which is present in both orientations in approximately equal amounts in the DNA population, and a...
Plasma oxytocin concentrations in cyclic mares and sexually aroused stallions. An experiment was conducted to measure plasma oxytocin concentrations at 4 different stages of the estrous cycle in 11 pony mares. Plasma oxytocin concentrations (muU/ml +/- SE) were found to be higher (P<.01) on day 2 of estrous (39.8 +/- 12.5) and day 5 post-ovulation (33.1 +/- 12.0) than on day 10 (2.3 +/- 1.6) and day 15 post-ovulation (6.8 +/- 4.1). A second experiment was conducted to measure jugular plasma oxytocin concentrations before and after sexual arousal in six pony stallions. Oxytocin concentrations (muU/ml +/- SE) were higher (P<0.06) after sexual arousal (50.5 +/- 8.9) than be...
Concentrations of progesterone, 17 alpha-hydroxyprogesterone and 20 alpha-dihydroprogesterone in the plasma of mares during pregnancy and at parturition. Plasma concentrations of progesterone and 17 alpha-hydroxyprogesterone were high in the 2nd and 3rd months of gestation, but 20 alpha-dihydroprogesterone increased from a level of 2 ng/ml, during the first 3 months, to 10-15 ng/ml during months 5-10, to reach 80-120 ng/ml during the last 30 days before foaling.
Collection and cultivation in vitro of equine mammary macrophages. Equine macrophages were obtained from female Shetland ponies by injection of Escherichia coli lipopolysaccharide through the lactiferous ducts of the mammary gland. After 6 to 11 days, balanced salt solution was injected into the mammary gland to wash out accumulated cells. Harvested cells contained a mixture of macrophages, lymphocytes, and neutrophils, with the majority of the cells of mononuclear type. In culture, cells adherent after 24 hours were characterized as macrophages by morphologic features, nonspecific esterase staining, and by the presence of complement and immunoglobulin recept...
Molecular cloning of equine herpesvirus type 1 DNA: analysis of standard and defective viral genomes and viral sequences in oncogenically transformed cells. Genomic DNA sequences of equine herpesvirus type 1 (EHV-1) have been cloned as BamHI and EcoRI restriction fragments into the plasmid pBR322 and propagated in Escherichia coli. With the exception of two EcoRI restriction fragments that reside in the S region of the viral genome, all of the cloned fragments demonstrated the same electrophoretic mobilities, restriction cleavage sites, and blot-hybridization patterns as did the parent fragments produced by BamHI or EcoRI digestion of virion DNA. The EcoRI J fragment and the BamHI E fragment of the L-region terminus were cloned after the addition ...
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...
