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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.
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 ...
Specificity of response to viral proteins in horses infected with equine infectious anemia virus. Three structural proteins of equine infectious anemia virus were purified, labeled with 125I, and utilized in radioimmunoassays with horse sera and antisera to heterologous retroviruses. Whereas radioimmunoassay titers for the major protein, p25, were 500- to 1,000-fold higher than titers in immunodiffusion, for clinical purposes these two procedures were equivalent. Antibodies to two low-molecular-weight proteins, p12 and p10, were also found in infected horses, but with a lower frequency and lower titers. As a rule, only sera positive for p25 also contained antibody to p12 and p10. Antisera ...
Lymphocyte specificity to protein antigens. II. Fine specificity of T-cell activation with cytochrome c and derived peptides as antigenic probes. Murine T-lymphocyte specificity was determined in a system of antigen driven in vitro T-cell proliferation using cytochrome c molecules from different species, their derived peptides and reconstituted hybrid proteins. It was observed that primed T cells could discriminate between peptide fragments which differed from each other at a single amino acid residue. These conclusions were substantiated by the pattern of cross-reactivity noted in the response of closely related cytochrome c proteins as well as when artificial hybrid molecules reconstituted by the covalent linkage of peptide fragments ...
Determination of the charge of horse kidney metallothionein by free boundary electrophoresis. Traditionally, the charge of a protein molecule as determined by electrophoresis has been compared to that revealed by pH titration, and any lack of coincidence has been ascribed to ion binding, and the two results have been brought into agreement by adjustment of binding parameters (1). Metallo-thionein allows a unique opportunity to examine the validity of the electrophoretic approach, since the amino acid sequence and metal atom binding studies allow the absolute charge of the molecule to be computed (2). This then can be compared to the charge determined from electrophoretic mobility measu...
Stability of the lyophilized F(ab’)2 fragments of horse tetanus antibodies isolated by affinity chromatography. F(ab')2 fragments of horse tetanus antibodies were obtained from horse hyperimmune sera after peptic digestion. The digest was passed through a column of tetanus toxoid coupled with Sepharose 4B, F(ab')2 fragments were eluted with a solution of 5 mM HCl in 150 mM NaCl and the eluates were concentrated by ultrafiltration and lyophilized. Glycine and human serum albumin were used as stabilizing agents. Polyacrylamide gel electrophoretic mobility and molecular weight of the fragments remained unchanged after lyophilization. Freeze-dried preparations stored two months at 56 degrees C showed only a...
An investigation of the uterine luminal environment of non-pregnant and pregnant pony mares. Uterine flushings were collected from 30 non-pregnant Pony mares on Days 8, 12, 14, 16, 18 or 20 after oculation. Mares were allowed a recovery period of one oestrous cycle and were mated at the next oestrus. They were then ovario-hysterectomized on days which corresponded to the day of the oestrous cycle to which they were assigned. Uterine flushings were analysed for total recoverable protein and acid phosphatase activity. Least squares analysis indicated a status X day interaction for total protein (P less than 0.10) and acid phosphatase activity (P less than 0.005) in which the latter was ...
The nature of the prealbumin ‘esterases’ of horse serum. Evidence is presented to suggest that the acidic prealbumin esterases in horse serum represent a protease-inhibitory protein. The esterase activity may arise from residual enzymic activity of the bound protease.
Contagious equine metritis–outbreak of the disease in Kentucky and laboratory methods for diagnosing the disease. Contagious Equine Metritis (CEM) was initially reported during the 1977 breeding season in England (Crowhurst, 1977) and Ireland (Timoney, Ward & Kelly, 1977. The disease has also been diagnosed in France and Australia (Huges, Bryden & MacDonald, 1978). The first occurrence of CEM in the United States followed the importation or 2 stallions from France late in 1977 which resulted in an outbreak early in the 1978 breeding season (Swerczek, 1978). Mares usually develop clinical signs of CEM 8--10 days after being covered by an infected stallion, when a copious, greyish discharge is seen. Other m...
An investigation of seven enzymes as possible genetic markers in horse leucocytes. In this paper we describe seven enzymes, NP, GOTM, PGM2, alpha FUC, PEP A, ADA and MPI which are found in the white cells of horses, including 39 British crossbred ponies and 16 crossbred horses, 30 Mongolian ponies and 10 Icelandic ponies. Two of these enzymes--alpha FUC and MPI--were polymorphic in all the populations of horses studied and could prove useful as additional markers in the paternity testing of horses. PEP A and GOTM were also polymorphic in two of the populations studied and could be used as further markers in these populations.
An enzyme immunoassay (EIA) for progesterone in horse plasma. A simple enzyme immunoassay (EIA) for the measurement of progesterone is described. Antibody against 11-OH-hemisuccinate-BSA is bound to polystyrene tubes. 11-OH-hemisuccinyl-beta-D-galactosidase is used as enzyme-coupled antigen and methylumbelliferyl-beta-D-galactoside as substrate. Concentrations down to 0.156 ng/ml plasm or amounts of 93 pg/tube are detectable. Probit analysis gave a linear relationship between log concentration and percentage of binding. A comparison of EIA and radioimmunoassay gave a correlation coefficient of 0.81. The assay is sufficiently sensitive to estimate progest...
