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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.
Isolation and characterization of antibodies to Clostridium perfringens epsilon toxin from hyperimmune horse serum. Antibodies against epsilon toxin were isolated from hyperimmune horse serum by affinity chromatography. Purified epsilon prototoxin covalently bound to Affigel 202 was used as immunosorbent, and antibodies were eluted with 6.0 M guanidine chloride. In a single run 80 mg of antibody could be recovered from a 20 microliter column of immunosorbent. The antibody was shown to belong to the IgG(T) class of immunoglobulins.
Occurrence of Leu-Lys-bradykinin and histidine-rich peptide in high-molecular-weight kininogen isolated from horse plasma. On incubation of purified horse plasma high-molecular-weight kininogen with purified plasma kallikrein, three new peptides, named fragment 1.2, fragment 1 and fragment 2, were released, in addition to the vasopeptide, bradykinin. Fragment 2 contained an extremely high level of histidine, in which eleven residues out of the total 48 residues were characterized. Thus the result proves the existence of the histidine-rich region in horse high-molecular-weight kininogen, which is similar to the region previously identified in bovine high-molecular-weight kininogen. Moreover, we have identified a ne...
Survival of contagious equine metritis bacteria in transport media. Survival of bacteria that cause contagious equine metritis (CEM) was evaluated in Amies modified transport (AMT) medium, in AMT medium with charcoal, and in Stuart transport medium at 37, 22, 4, and -70 C. The CEM bacteria suspended in transport media survived at 22, 4, and -70 C for longer periods in AMT medium with charcoal than they did in AMT and Stuart transport media. In 1 day, the number of bacteria in exudate stored in the absence of any transport medium decreased 15-fold at 22 C and twofold at 4 C. The CEM bacteria were isolated from exudate on cotton-tipped swabs from all three trans...
Purification of the subunit Clq from the first component of equine complement. Initial separation and concentration of Clq from fresh, normal equine serum was accomplished by precipitation in 0.02 M acetate buffer, pH 5.5, at 4 degrees for 24 h. The re-dissolved precipitate was clarified by centrifugation at 80,000 g for 1 h and then dialysed against Tris-HCl buffer (0.05 M, pH 8.0) containing 10-3 M EDTA. The clarified dialysate remained biologically active at 5 degrees for at least 4 weeks. Biological activity of equine Clq was determined by assay of its ability to agglutinate sensitized sheep erythrocytes (EA). Following ammonium sulphate fractionation, Sepharose 4B g...
In vitro of adenosine on lymphocytes and erythrocytes from horses with combined immunodeficiency. The effect of adenosine on the mitogenic response of peripheral blood lymphocytes (PBL) and on the nucleotide pools of erythrocytes from normal horses, horses heterozygous for the combined immunodeficiency (CID) trait (carriers), and foals with CID was studied. When PBL from normal, carrier, and CID horses were stimulated by phytohemagglutinin (PHA), concanavalin A, or pokeweed mitogen, [3H]thymidine uptake was inhibited by adenosine (0.1 microM) to 1.0 mM) in a dose-dependent manner. Adenosine (100 microM) mediated inhibition of [3H]thymidine uptake was prevented in both normal and carrier ho...
Collagenase in equine cell culture preparation. Equine kidney cells disaggregated by treatment with 0.01% collagenase were used in the preparation of primary monolayer cell cultures. The primary cells could be stored for long periods in liquid nitrogen and subsequently subcultivated. These techniques provided a long-term supply of equine kidney cells, free of apparent contamination, from the kidneys of a single fetus.
Immunochemical studies on beta-lactoglobulins. precipitin reactions of sow’s and mare’s mammary secretions against anti – bovine beta – lactoglobulin antiserum. By double diffusion in agarose gel, in well defined experimental conditions, cross reactions were observed between porcine beta-lactoglobulins and anti-bovine beta-lactoglobulin antisera. The immunological reactivity between these beta-lactoglobulins from a monogastric and the ruminant anti beta-lactoglobulin antiserum thus implies a certain degree of similarity between the monomeric beta-lactoglobulins examined and the dimeric of the ruminants. With the same antisera it also proved possible to demonstrate the presence of beta-lactoglobulins in the mammary secretions of another monogastric, na...
Persistent infection of a human lymphoblastoid cell line with equine herpesvirus 1. Infection of a human lymphoblastoid cell line (Jijoye line derived from a Burkitt lymphoma which contains Epstein-Barr virus) with equine herpesvirus 1, maintained and observed for 53 days, was characterized by the continuous production of infectious extracellular and intracellular virus. Maximum virus production correlated with active cell multiplication. Less than 15% of the cells possessed viral capsid antigen at any one time. Five percent of the cells in the Jijoye line possess Epstein-Barr viral capsid antigen; 80% of the Epstein-Barr viral caspid-containing cells also contained equine he...
Haptoglobin in the serum of thoroughbreds in training. A method is described for the measurement of haptoglobin in equine serum using the peroxidase activity of the haemoglobin-haptoglobin complex. The problems of interference with Fe2+ and Fe3+ ions are described. Normal values for haptoglobin in 629 blood samples from thoroughbreds in training are presented showing a log normal distribution with a 5 per cent to 95 per cent range of 0.42 to 1.7 g/litre. There was no consistent alteration in haptoglobin concentration throughout the season in spite of a change in red cell size and total bilirubin concentration. It is concluded that the measurement ...
Selective medium for Corynebacterium equi isolation. The development of a selective medium for the isolation of Corynebacterium equi is described. The medium has been used to examine fecal samples from 127 horses of which 90 have been found to carry the organism.
The effect of binding ions on the oxidation of horse heart ferrocytochrome c. The research explores how different binding ions affect the oxidation speed of horse heart ferrocytochrome c, a protein, by potassium ferricyanide at a constant ionic strength. Studying the Ion Effect […]
Molecular cytogenetics of the Equidae. I. Purification and cytological localization of a (G + C)-rich satellite DNA from Equus przewalskii. A (G + C)-rich density satellite DNA (rho = 1.713 gm/cc) has been purified from splenic DNA of Przewalski's horse, Equus przewalskii, by successive equilibrium density gradient centrifugations. The purified satellite, which may comprise as much as 29% of the total DNA, renatures rapidly; however, analyses of native, single-stranded, and reassociated molecules by analytical ultracentrifugation and melting properties suggest that some sequence heterogeniety exists in the 1.713 gm/cc satellite. Complementary RNA (cRNA) transcribed from satellite DNA has been utilized for in situ hybridization stu...
Chronic nephritis in a pony. The clinical and pathological features of a case of chronic nephritis in a 17-year-old pony was described. Measurement of fluid intake and laboratory analysis of sequential blood and urine samples helped in establishing an accurate diagnosis. The case demonstrates that although chronic renal disease is not well documented in the horse it should nevertheless be considered in the differential diagnosis of conditions characterised by progressive loss of weight.
Possible modification of scar tissue by biochemical methods. This paper reviews some of the biochemical modifications involved in fibrous tissue formation and discusses possible ways of controlling fibrosis in clinical conditions. The lathyritic agents, beta-aminoproprionitrile (BAPN) and penicillamine, appear in certain situations to be able to control fibrosis by blocking the biosynthesis of collagen. There are no compounds that are yet known which are capable of reversing pre-existing fibrosis and future research may perhaps be more profitably directed towards the stimulation of collagen catabolism rather than the inhibition of its synthesis.
Comparative serologic study of equine piroplasmosis, with card and complement-fixation tests. An agglutinating antigen and a rapid card test (CT) for equine piroplasmosis was developed. The antigen for the CT was prepared from lyophilized Babesia caballi complement-fixation (CF) antigen. Serum and plasma samples for testing were obtained from known B caballi-infected horses and clinically normal horses maintained at the laboratory. Serum samples also were obtained from horses outside the continental United States, in areas where piroplasmosis is endemic. Comparative CT and CF tests were done on all samples. The CT correctly identified 85% of 192 plasma samples from known infected and n...
A passive haemagglutination test for the detection of antibodies to the contagious equine metritis organism. A passive haemagglutination test (PHT) which has been developed for the detection of antibodies to the contagious equine metritis organism (CEMO) in serum is described. Samples from each of 30 mares with metritis were positive with titres in the range 256 to 4096. Samples from each of 239 clinically normal mares and 30 colts and fillies believed not to have been exposed to CEMO were negative with titres of less than 256, the majority of samples (97 per cent) showing a titre of 32 or less.
A mechanistic model for butyrylcholinesterase. A plausible mechanism of action of horse serum butyrylcholinesterase is proposed. It includes substrate activation at the level of deacylation. The rate constant for the acylation of the enzyme appears to be much greater than the rate constant for the deacylation, at low substate concentrations. At higher substrate concentrations the rate constants become more similar. No interaction between the four subunits in binding of inhibitors or in the catalysis was observed. There is one esteratic and one anionic site per subunit apparent from labelling studies with [32P]diisopropylfluorophosphate and...
Insensitivity of the ferritin iron core to heat treatment. To test whether the reactivity of ferritin iron is affected by the heat treatment used in ferritin isolation, we prepared ferritin from the same horse spleen with or without heating. Both samples exhibited similar reactivity upon reduction or chelation of iron.
Contagious equine metritis: a review. Contagious Equine Metritis (CEM) is a highly contagious venereal disease of horses caused by a fastidious, Gram-negative coccobacillus which grows best on chocolate agar under microaerophilic conditions (5-10% CO2). Clinically, the disease is characterized by a copious watery-to-mucopurulent, vaginal discharge two to ten days after breeding by an infected stallion (11, 13). Shortened estrous cycle lengths are common and may be the only indication of endometritis in some instances (7). Inapparent carriers of the disease in both the mare and stallion make control of the disease more difficult. O...
Synthesis of long complementary DNA in the endogenous reaction by equine infectious anemia virus. In the endogenous reverse transcriptase reaction, equine infectious anemia virus is able to synthesize complementary DNA (cDNA) of 8,000 nucleotides in high yield. After 2 h in 50 muM dNTP, about 2.8 mug of cDNA per mg of protein is produced, almost 30% of which is long cDNA. The system thus compares favorably with the other two well-characterized endogenous reaction systems, Moloney murine leukemia virus and avian sarcoma virus. Elongation rates of 100 to 150 nucleotides per min have been observed; these rates are comparable to those seen with purified avian myeloblastosis virus reverse trans...
Radioimmunoassay for PMSG and its application to in-vivo studies. A double-antibody radioimmunoassay for PMSG, especially for meauring PMSG in cattle blood after exogenous application, has been developed. A rabbit antiserum against PMSG and pure PMSG for radioiodination were used. There was a strong cross-reaction against equine LH and FSH, but the slight cross-reaction against bovine LH and FSH could be eliminated by adding bovine LH to each tube during the assay. Unspecific, interfering influences of equine or cow serum could be eliminated by adding a constant amount of PMSG-free serum to each tube. PMSG added to 200 microliter of serum could be recovered ...
