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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.
Monoclonal antibodies to the equine CD2 T lymphocyte marker, to a pan-granulocyte/monocyte marker and to a unique pan-B lymphocyte marker. Murine monoclonal antibodies, HB88A, B29A and DH59B separately identify the CD2 T lymphocyte molecule, a unique pan-B lymphocyte surface marker and a pan-granulocyte/monocyte surface molecule, respectively, in the horse. Specificity was shown by two-color immunofluorescent flow cytometry and immunofluorescent microscopy. MAb HB88A reacted with a 52 kDa pan-T lymphocyte molecule present on 75% +/- 7 of peripheral blood lymphocytes (PBL) (n = 15 horses). It also reacted with lymphocytes restricted to T lymphocyte dependent areas of lymph node and spleen. Specificity of mAb HB88A to CD2 was demon...
Pentoxifylline inhibits mediator synthesis in an equine in vitro whole blood model of endotoxemia. Whole blood from 10 healthy horses was aseptically collected into heparin or citrate anticoagulant and incubated in vitro for 6 hr in the absence (saline control) or presence of 1 ng endotoxin/ml blood. Pentoxifylline (0.1, 1, 10, or 100 micrograms/ml blood) was added 1 hr before, at the same time, or 1 hr after endotoxin. As compared to saline controls, pentoxifylline alone had no effect on mediator production, with the exception of significantly increasing 6-ketoprostaglandin F1 alpha concentration. Pentoxifylline inhibited endotoxin-induced increases in tumor necrosis factor (TNF) and inter...
Endogenic nortestosterone in cattle? When residues of nortestosterone (NT) were found in the urine of cattle, racehorses or bodybuilders, exogenic administration was thought to be proven. In previous literature, no records were found of the endogenic presence of this molecule. In the horse-racing world, Houghton and Courthot found that NT is normally present in the urine of the stallion. Belgian and Dutch researchers found that NT is also present in the urine and edible parts of the intact boar. Vandenbroeck et al. (1991) suggested the endogenous presence of NT (in the beta form) in the pregnant cow. Meyer (1992) reported the pre...
Determination of free malonaldehyde formed in liver microsomes upon CCl4 oxidation. Free malonaldehyde formed in the microsomes prepared from livers of monkey, rat, rabbit, mouse, cow, pig, dog, sheep and horse upon CCl4 oxidation was derivatized by reaction with N-methylhydrazine to form 1-methylpyrazole which was subsequently analyzed by capillary gas chromatography. Among the livers from animals tested, the monkey and rat livers produced the most malonaldehyde upon CCl4 treatment. Horse liver showed the greatest resistance to CCl4 oxidation. The gas chromatography method used in the present study exhibited an accurate and specific measurement of free malonaldehyde that mig...
Insulin-like growth factor II in the horse: determination of a cDNA nucleotide sequence and expression in fetal and adult tissue. Horse cDNA for insulin-like growth factor II (IGF II) has been isolated. cDNA was synthesized from bulk mRNA and subsequently PCR-amplified and sequenced. Like its human counterpart, the mature horse IGF II peptide contains 67 amino acids with only two substitutions, isoleucine instead of valine in position 35 and asparagine instead of serine in position 36. The nucleotide homology was 92.1% between horse and human and 87.8% between horse and mouse. The isolated cDNA hybridized to multiple transcripts in fetal and adult tissues, thus confirming earlier reports on developmental expression of th...
Effect of sample handling on measurement of plasma glucose and blood lactate concentrations in horses before and after exercise. Collection of a satisfactory blood sample requires special procedures to prevent changes in glucose and lactate content after the sample has been obtained. Changes in measured plasma glucose and blood lactate concentrations attributable to anticoagulants and storage procedures, respectively, were examined in blood samples obtained from horses at rest and after exercise. To evaluate the effect of anticoagulants on measured plasma glucose concentration, blood was preserved with either sodium fluoride/potassium oxalate or lithium heparin. Measured plasma glucose concentration in blood obtained at...
GRASP: a novel heparin-binding serum glycoprotein that mediates oligodendrocyte-substratum adhesion. Cell-substratum adhesion plays a crucial part in the cascade of events that control growth or turn on and consummate a differentiation program. We are investigating the molecular basis of oligodendrocyte (OLG) cytodifferentiation, employing pure cultures of OLGs isolated from postmyelination brains. We have shown that such OLGs will regenerate in vitro and reenact the ontogenic development of myelin, but to do so they need a signal. Adherence to a polylysine surface in the presence of 20% horse serum generates such a signal. Among the events that are turned on upon OLG adhesion is the phosphor...
Equilibrium unfolding studies of horse muscle acylphosphatase. The stability and equilibrium unfolding behaviour of horse muscle acylphosphatase have been studied by denaturing the protein under various conditions of temperature, pH, and urea concentration. Far-ultraviolet circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy indicate that this small monomeric protein unfolds reversibly and cooperatively. Thermodynamic parameters, the Gibbs free energy delta G and enthalpy delta H of unfolding, have been estimated for denaturation of the protein from NMR and CD data as 19 kJ mol-1 and 350 kJ mol-1, respectively. CD and 1H-NMR results s...
Studies on the presence of magnesium in visceral amyloid. The Magneson and Titan yellow tests were used to detect Mg in amyloid deposits in primary and secondary amyloidosis, in a pituitary tumour and in a case of equine cutaneous amyloidosis. Especially vascular amyloid deposits turned out to contain high levels of Mg. The significance of these findings remains unclear at present. Competition between Mg and Ca, the relationship between Mg in elastic fibres and amyloid P-component, and the high water content of amyloid along with the interaction of Mg with water are discussed.
Structural protein relationships among eastern equine encephalitis viruses. We have re-evaluated the relationships among the polypeptides of eastern equine encephalitis (EEE) viruses using SDS-PAGE and peptide mapping of individual virion proteins. Four to five distinct polypeptide bands were detected upon SDS-PAGE analysis of viruses: the E1, E2 and C proteins normally associated with alphavirus virions, as well as an additional more rapidly-migrating E2-associated protein and a high M(r) (HMW) protein. In contrast with previous findings by others, the electrophoretic profiles of the virion proteins of EEE viruses displayed a marked correlation with serotype. The pro...
The complete mitochondrial DNA sequence of the horse, Equus caballus: extensive heteroplasmy of the control region. The sequence of the mitochondrial (mt) DNA of the horse (Equus caballus) was determined. The length of the sequence presented is 16,660 bp. This figure, however, is not absolute due to pronounced heteroplasmy caused by variable numbers of the motif GTGCACCT in the control region of different molecules. Boundaries of the 13 peptide-coding genes were determined by the presence of start and stop codons, and by analogy with other eutherian mtDNAs. Three genes (COIII, NADH3 and NADH4) were not terminated by a stop codon. Comparison among the peptide-coding genes of the horse and eight other mammals...
Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms. A new method for typing single nucleotide polymorphisms in DNA is described. In this method, specific fragments of genomic DNA containing the polymorphic site(s) are first amplified by the polymerase chain reaction (PCR) using one regular and one phosphorothioate-modified primer. The double-stranded PCR product is rendered single-stranded by treatment with the enzyme T7 gene 6 exonuclease, and captured onto individual wells of a 96 well polystyrene plate by hybridization to an immobilized oligonucleotide primer. This primer is designed to hybridize to the single-stranded target DNA immediately...
In vitro fertilization rate of horse oocytes with partially removed zonae. Frozen-thawed ejaculated stallion spermatozoa were preincubated for 3 h in BO medium containing 5 mM caffeine and then treated with 0.1 micro M calcium ionophore A23187 for 60 sec. Aliquots of the sperm suspension (final concentration 1-2 x 10(7)/ml) were added to the oocytes which had been matured in vitro for 32 h. In Experiment 1, there were 3 groups of oocytes; cumulus intact, denuded zona-intact, and zona-free. Cumulus cells were removed with 0.5% hyaluronidase and the zona pellucida with 0.1% protease. The oocytes were fixed 20 h after insemination with acetic acid:ethanol (1:3) and stai...
Decrease in the alpha 1-proteinase inhibitor Spi3 in equine bronchoalveolar lavage fluid. The alpha 1-proteinase inhibitors of trypsin, Spi1, Spi3A, and Spi3B, in bronchoalveolar lavage fluid (BALF) and serum of horses were separated by electrophoresis, and their proportions were quantified in 12 control horses and 12 with chronic obstructive pulmonary disease (COPD). A significantly lower proportion of Spi3B (P < 0.05) and higher proportion of Spi1 (P < 0.02 to P < 0.01) were detected in BALF, compared with serum, in control and COPD-affected horses and appeared to be attributable to reduced Spi3 activity in BALF. There was no significant difference between the control an...
Monocyte maturation controls expression of equine infectious anemia virus. In vivo, equine infectious anemia virus (EIAV) replicates in tissues rich in macrophages, and it is widely believed that the tissue macrophage is the principal, if not sole, cell within the host that replicates virus. No viral replication has been detected in circulating peripheral blood monocytes. However, proviral DNA can be detected in these cells, and monocytes may serve as a reservoir for the virus. In this study, an in vitro model was developed to clarify the role of monocyte maturation in regulating EIAV expression. Freshly isolated, nonadherent equine peripheral blood monocytes were in...
Characterization of a novel streptokinase produced by Streptococcus equisimilis of non-human origin. Streptokinases are proteins with plasminogen activator activity produced by certain hemolytic streptococci. We previously identified equine streptococcal isolates which produced streptokinases (ESKs) that bound both human and equine plasminogen but only readily activated equine plasminogen (14). This property was exploited to purify a representative ESK produced by Streptococcus equisimilis strain 87-542-W. Affinity chromatography with human plasminogen resulted in the isolation of a M(r) approximately 49,000 molecule with two isoforms. This ESK was subsequently compared to well characterized ...
In vitro susceptibility of equine Salmonella strains to trimethoprim and sulfonamide alone or in combination. The in vitro activity of trimethoprim (TMP) and 9 sulfonamides and their combinations in 6 concentration ratios was tested against 62 Salmonella strains isolated from horses over a 3-year period in the Netherlands, using the agar-dilution method. Most of the isolates were S typhimurium strains (n = 52); the others were S heidelberg (n = 3), S hadar (n = 2), S thompson (n = 2), S enteritidis (n = 1), S infantis (n = 1), and S derby (n = 1). The minimal TMP concentration at which 50% of the Salmonella strains were inhibited (MIC50) was 0.12 micrograms/ml. Sulfachlorpyridazine (SCP; MIC50, 16 mic...
Further characterisation of forms of haemosiderin in iron-overloaded tissues. The biochemical and biophysical properties of isolated haemosiderins have been compared to that of another iron-containing protein, termed prehaemosiderin, which sediments through chaotropic potassium iodide only after 20 h of ultracentrifugation, in contrast to that of haemosiderin which is recovered after 2 h of ultracentrifugation. The iron/protein ratio and iron/phosphate ratio were less that that of the corresponding haemosiderin, while the elemental composition was also reduced in many of the prehaemosiderin samples. Mossbauer spectroscopy and electron diffraction identified the predomin...
Rapid diagnosis of equine influenza by the Directigen FLU-A enzyme immunoassay. The Directigen FLU-A enzyme immunoassay was tested for its ability to detect equine-2 influenza viruses in nasopharyngeal fluids from horses and ponies. A total of 125 swabs from experimental infections and from different sources of natural infection in the USA and Hong Kong were examined. The assay results were compared with the results of standard virus culture in embryonated chicken eggs or Madin-Darby canine kidney cells, and with the serology of the horses sampled. In comparison with virus culture the enzyme immunoassay exhibited 83 per cent sensitivity, 78 per cent specificity, 70 per ce...
Detection of Ehrlichia risticii from feces of infected horses by immunomagnetic separation and PCR. Potomac horse fever, caused by Ehrlichia risticii, is an important disease of equines. The major features of the disease are fever, leukopenia, and diarrhea. The organism has been detected from the blood mononuclear cells of infected horses, but its presence in the feces has not been known. A method for immunomagnetic separation of E. risticii from the feces of infected horses was developed, and the separated organisms were detected by PCR. Coating immunomagnetic beads (Dynabeads) with a 1:5 dilution of rabbit anti-E. risticii serum and incubating the Dynabeads with fecal samples for 25 min at...
A new type of staphylococcal exfoliative toxin from a Staphylococcus aureus strain isolated from a horse with phlegmon. A new type of staphylococcal exfoliative toxin (sET) was isolated from the culture filtrate of a Staphylococcus aureus strain isolated from a horse with skin infection including phlegmon. The new sET was purified by precipitation with 80% saturated ammonium sulfate, column chromatography on DEAE-cellulofine A-500, gel filtration on a Sephadex G-75 column, and polyacrylamide gel electrophoresis (7.5% polyacrylamide). The new sET elicited general exfoliation of the epidermis with the so-called Nikolsky sign when inoculated into both 3-day-old mice and 1-day-old chicks, whereas sETA and sETB from...
Proteins induced by recombinant equine interferon-beta 1 within equine peripheral blood mononuclear cells and polymorphonuclear neutrophilic granulocytes. Peripheral blood mononuclear cells (PBMC) and polymorphonuclear neutrophilic granulocytes (PMN) as well as embryonic equine dermal fibroblasts and the equine fibroblast line E. Derm which were used as controls, were treated with recombinant equine interferon-beta 1 (rEqIFN-beta 1) in vitro which induced the expression of different proteins in these cells. A 74 kDa protein was induced in PBMC and an 82 kDa protein was additionally found in the equine fibroblast E. Derm cell line following treatment with rEqFN-beta 1. Both proteins reacted with anti-mouse and anti-human Mx protein antisera in im...
Monoclonal antibodies to equine arteritis virus proteins identify the GL protein as a target for virus neutralization. Monoclonal antibodies (MAbs) to equine arteritis virus (EAV) proteins were produced and characterized. The protein specificities of eight MAbs were determined definitively by immunoprecipitation of EAV proteins expressed from vaccinia virus recombinants (VVRs). Included were two new VVRs produced for this study, expressing the M and the GL proteins, respectively. Three MAbs were determined to be N-specific and five MAbs recognized the GL protein. One GL-specific MAb, 17F5, of the IgA class, efficiently neutralized EAV infectivity. In competitive binding assays (CBAs), the N-specific MAbs defin...
Proteolytic processing of the replicase ORF1a protein of equine arteritis virus. To study the proteolytic processing of the equine arteritis virus (EAV) replicase open reading frame 1a (ORF1a) protein, specific antisera were raised in rabbits, with six synthetic peptides and a bacterial fusion protein as antigens. The processing of the EAV ORF1a product in infected cells was analyzed with Western blot (immunoblot) and immunoprecipitation techniques. Additional information was obtained from transient expression of ORF1a cDNA constructs. The 187-kDa ORF1a protein was found to be subject to at least five proteolytic cleavages. The processing scheme, which covers the entire OR...
Potency of partially purified malleo-proteins for mallein test in the diagnosis of glanders in equines. Malleo-proteins from synthetic broth mallein of six strains of Pseudomonas mallei were separated by trichloroacetic acid precipitation, ammonium sulfate precipitation and Ultrogel AcA 34 gel filtration chromatography. When tested comparatively with Dutch PPD mallein as standard on P. mallei-sensitized and normal horses all the strains were found to be malleinogenic, trichloroacetic acid precipitated proteins were comparable to Dutch PPD mallein in potency and innocuity whereas ammonium sulfate-precipitated proteins elicited non-specific reactions. Ultrogel AcA 34 chromatographed high molecular...
Molecular cloning and expression of two horse pancreatic cDNA encoding colipase A and B. Pancreatic colipase plays an essential role in the intestinal fat digestion by anchoring lipase on lipid/water interfaces in the presence of bile salts. In contrast to other species, two molecular forms of colipase, A and B, have been found in horse. The two corresponding cDNAs were isolated from a horse pancreatic library and their nucleotide sequences were determined. Moreover, for the first time, active colipase has been obtained after transfection of COS cells by either colipase A or B cDNA.
Diagnosis of African horsesickness. African horsesickness (AHS) is a very serious, non-contagious disease of horses and other solipeds caused by an arthropod-borne orbivirus of the family Reoviridae. The epizootic nature of the disease makes rapid, accurate diagnosis of AHS absolutely essential. Currently, diagnosis of AHS is based on typical clinical signs and lesions, a history consistent with vector transmission and confirmation by laboratory detection of virus and/or anti-AHS virus antibodies. The clinicopathologic presentation of AHS, current and next generation laboratory diagnostic methods are discussed.
