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.
Ushimi C, Henson JB, Gorham JR.Primary horse leukocyte cultures were inoculated with 2 or 10 50% tissue culture infective doses (TCID(50)) of equine infectious anemia (EIA) virus per cell, and the titer of cell-associated and fluid-phase virus was determined from 1 to 72 hr postinoculation (PI). Cover slips were collected from 4 to 72 hr PI and stained for EIA viral antigen by the indirect immunofluorescent (FA) technique. Viral replication was detected after a latent period of approximately 18 to 24 hr and reached peak titers of approximately 10(4.5) to 10(6) TCID(50)/0.5 ml from 48 to 72 hr PI. The fluid phase contained 1...
Brunel C, Cathala G.1. Alkaline phosphatases (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) from brain, kidney, liver, bone, lung and spleen, which are not very sensitive to l-phenylalanine, are strongly inhibited by imidazole, whereas the placental and intestinal enzymes, which are very sensitive to l-phenylalanine, are only slightly affected. This is a new possibility for distinguishing the alkaline phosphatase isoenzymes.
2. The inhibition is apparently of an uncompetitive type, suggesting that the inhibitor interacts with the ES complex to form an EIS complex.
3. Histidine acts upon all enzyme...
Barron AL, Caste PG, Paul B, Page LA.Postinoculation sera collected from pigeons, turkeys, guinea pigs, sheep, a calf, a rabbit, and a horse experimentally infected with various strains of Chlamydia psittaci yielded a high incidence of positive reactions when tested by double diffusion in gel. Antigen was a deoxycholate extract of SA-2 strain of C. trachomatis. Good correlation was obtained with results of complement fixation tests, whereas double diffusion in gel was less sensitive. Immunoelectrophoresis of the antigen revealed presence of two antigens in the extract.
Boulanger P, Bannister GL, Carrier SP.An agar-gel immunodiffusion test recommended for the diagnosis of equine infectious anemia was evaluated. Our preliminary observations confirmed those of Coggins concerning the mechanism of the test and the results obtained. Furthermore, emphasis was put on the difficulties encountered in the production of spleen antigens with an optimum amount of reactivity. Acetone-ether extraction procedures for the preparation of a liquid antigen extract are described. This type of antigen was reactive in the complement-fixation test in 1:8 or greater dilution and it is proposed to use the complement-fixat...
Schams D, Papkoff H.Highly purified pregnant mare serum gonadotropin (PMSG) can be prepared from crude commercial preparations of PMSG by chromatography on sulfoethyl-Sephadex C-50 and gel filtration on Sephadex G-100. The preparation was examined by disc electrophoresis and gel filtration and found to be of high purity. Amino acid analysis shows similarities to pituitary gonadotropins. The PMSG contains a high content of proline and cystine and low amounts of the aromatic amino acids. Phenylalanine is the major amino terminal amino acid. The carbohydrate content totals 45% of which 10% is the content of sialic a...
McManus AT, Robinson DM.Reconstituted Venezulean equine encephalitis vaccine was found to retain significant titers of plaque-forming virus after storage at 4 or 22 C for 24 hr.
Momose A, Tsuji T.When sulpyrine and aminopyrine are administered to the horse, unchanged aminopyrine and its metabolites, 4-methylaminoantipyrine and 4-aminoantipyrine, are detected in the urine by means of thin-layer chromatography (TLC) and gas liquid chromatography. Further identification of aminopyrine and these metabolites was carried out by the gas chromatography-mass spectrometry (GC-MS) method. The procedures for separation and identification are as follows : The excretions were adjusted to pH 9 with ammonium hydroxide and extracted with chloroform. The extract was separated by TLC. The spots were loca...
Butudom P, Foreman JH, Kline KH, Whittem EL.Some methods of lactate (LA) measurement have not been validated appropriately for use in horses. Objective: To validate 2 LA analysers (YSI 2300 Stat Plus and TDx Lactic Acid Assay) for use with equine plasma and to compare plasma [LA] determined by the 2 methods. Methods: Both instruments were evaluated for linearity, parallelism, recovery and precision using serial dilutions of standard LA solutions and equine plasma and then comparing results with linear regression or paired t tests. Plasma [LA] results were compared in 275 blood samples collected from horses exercising at various intensit...
Dillon AM, Heath MF.Protein tyrosine phosphorylation (PTP) in thrombin- and platelet-activating factor (PAF)-stimulated equine platelet activation was investigated in the absence and presence of 2 protein tyrosine kinase inhibitors (PTKIs), methyl 2,5-dihydroxycinnamate (MDHC) and genistein. Washed equine platelets aggregated irreversibly in response to thrombin or PAF in an agonist concentration dependent fashion. MDHC produced an MDHC concentration and time dependent inhibitory effect on rate and extent of thrombin- and PAF-induced aggregations, whereas the effect of genistein on the same parameters was only ge...
Yu DT, Gale RP, Kacena A, Pearson CM.Rosette formation between human lymphocytes and horse red blood cells could be promoted by a low pH medium, overnight incubation and a temperature of 4 degrees C. The percent of sheep, horse and human rosette-forming cells in the peripheral blood were 71.7 +/- 1.8, 30.5 +/- 2.8 and 28.3 +/- 3.4 respectively. However, their percentages in thymuses were 97.1 +/- 1.1, 91.4 +/- 2.4 and 89.0 +/- 3.4. Using preparations of isolated subpopulations, it was observed that the horse and human red cell rosette-forming cells were probably also "early" sheep red cell rosette-forming cells. Rosette formation...
Horteloup MP, Threlfall WR, Funk JA.The horse early conception factor (ECF) test is designed for qualitative determination of the ECF glycoprotein in the mare that has conceived. The objectives of this study were to determine the performance of the horse ECF test for the detection of the non-pregnant mare, and to determine the agreement among subjects or "readers" regarding the interpretation of the test. Blood samples from 60 mares were collected on Days 0, 5, 8, 11 and 18 following ovulation. Pregnancy status diagnosed with the ECF test was compared (2 x 2 table) to pregnancy status diagnosed by palpation per rectum and ultras...
Nakao T, Ohno-Fujitani T, Nakao M.Approximate molecular weights and the subunit structures of Na,K-ATPase from horse kidney were estimated by means of the combination of porous silica gel chromatography, laser light scattering (LS) and refractive index (RI) measurements in C12E8. When the enzymes were eluted with NaCl- or KCl-containing solution, 3 or 4 protein peaks, respectively were detected except that of low molecular weight range. These peaks were tentatively named Na-1, Na-2, Na-2', Na-3 (NaCl-containing eluents), K-1, K-2, K-3 (KCl-containing eluents), respectively. Na,K-ATPase and K-p-nitrophenylphosphatase activities...
Levine RA, Hart AH, Wardlaw SC.Using quantitative buffy coat analysis (QBCA), rapid and accurate measurements can be made of the erythrocyte PCV, total WBC count, and platelet count, and the leukocyte population can be differentiated into total granulocytes (including quantitation of eosinophils), and lymphocytes and monocytes. The QBCA is performed by placing a blood sample (50 to 111 microliters) into a high-precision-bore microhematocrit tube that contains a freely moving, closely fitting, cylindrical plastic float. After centrifugation for 5 minutes, the buffy coat components separate by density. The plastic cylinder fl...
Reilly CA, Aust SD.An intracellular, membrane-bound enzyme exhibiting both p-phenylenediamine oxidase activity and ferrous iron oxidase activity was isolated with the plasma membrane fraction of horse heart and studied for its ability to load iron into ferritin. The ferroxidase activity of the tissue oxidase was stimulated approximately twofold by horse spleen apoferritin, and the iron was loaded into ferritin. The loading of iron into ferritin by the tissue oxidase was inhibited by anti-horse serum ceruloplasmin antibody. The stoichiometry of iron oxidation and oxygen consumption during iron loading into ferrit...
Liang CZ, Cao RB, Wei JC, Zhu LH, Chen PY.According to the antigenic analysis of equine arteritis virus (EAV) GL protein, one pair of primers were designed, with which the gene fragment coding the high antigenic domain of EAV GL protein was amplified from the EAV genome. The cloned gene was digested with BamH I and Xho I and then inserted into pET-32a and resulted pET-GL1. The pET-GL1 was transformed into the host cell BL21(DE3) and the expression was optimized including cultivation temperature and concentration of IPTG. The aim protein was highly expressed and the obtained recombinant protein manifested well reactiongenicity as was c...
McDowell KJ, Adams MH, Williams NM.Proteins synthesized and released in vitro by oviducts collected from horse mares during estrus and at day 4 after ovulation for bred and nonbred mares were examined by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS PAGE) and fluorography. Ampullary and isthmic regions both produced a wide array of nondialyzable proteins in culture. Major proteins or groups of proteins identified according to relative molecular weight (kDa) and apparent isoelectric point (pI) were at 100 kDa, pI 8; 100-200 kDa, pI 6; 150 kDa, pI 4.5; 60-100 kDa, pI 4; and an array of polypep...
Bai Y, Tong T, Liu G, Chen W, Zhang W, Wang Q, Yang T, Bu Z, Wu D.Interferon gamma (IFN-gamma) is a pleiotropic cytokine that is recognized as an important modulator of the immune response. To date, there is no report that prokaryocyte-derived recombinant equine IFN-gamma has antiviral activity. In this report, the gene coding equine IFN-gamma (EIFN-gamma) mature protein was cloned into pET-28a (+) and the recombinant EIFN-gamma was expressed in Escherichia coli (E. coli). The antiviral activity of expressed recombinant EIFN-gamma was evaluated by using a recombinant Vesicular Stomatitis Virus expressing green fluorescence protein (rVSV-GFP) system in the eq...
Camargo FC, Robinson NE, Dirikolu L, Berney C, Eberhart S, Derksen FJ, Lehner AF, May J, Hughes C, Tobin T.Trimetoquinol (TMQ) is a very potent and fast acting bronchodilator in horses with heaves. This study assessed the plasma and urinary concentrations of TMQ in horses with heaves following administration via the intravenous (IV, 0.2 microg/kg) and intra-tracheal (IT, 2 microg/kg) routes. TMQ was administered to six horses affected with heaves (RAO - Recurrent Airway Obstruction, used interchangeably) by the above routes and plasma and urine samples collected and stored at -20 degrees C until analyzed. Solid Phase Extraction (SPE) of TMQ was followed by highly sensitive ESI(+)-LC-MS-MS (ElectroS...
Krumrych W, Skórzewski R, Malinowski E.The aim of this study was to determine the effect of duration and temperature of sample storage on whole blood chemiluminescence measurement results. Venous blood from 18 clinically healthy Polish half-bred horses aged 4 to 11 years were used in the study. Luminol dependent chemiluminescence (CL) was used to measure neutrophil oxygen metabolism in whole blood. Blood samples were examined for spontaneous CL and stimulated by a surface receptor stimulus as well as extra-receptor stimulus. The assay was performed in two parallel experimental sets with samples stored at 4 and 22 °C, respectively....
Stanley SD, McKemie D, Skinner W.A rapid, sensitive, and rugged method for detecting drugs and drug metabolites in extracts of horse urine is described. The use of large-volume injection (LVI) gas chromatography-mass spectrometry (GC-MS) for analysis of horse urine extracts allowed automation of the derivatization procedure and reduction of the sample volume from 5 mL to 1 mL of urine. An autosampler and temperature-programmable inlet were used to automatically dissolve the sample extract and form trimethylsilyl derivatives of over 200 analytes. The suitability of this procedure for routine GC-MS detection of approximately 80...
Singh AK, Gordon B, Hewetson D, Granley K, Ashraf M, Mishra U, Dombrovskis D.Gas chromatography with chemical ionization mass spectrometry and selected-ion monitoring provided a sensitive method for the screening and confirmation of steroids in horse urine and plasma. Chemical ionization mass spectrometry was more sensitive than the electron impact ionization mass spectrometry for most of the steroids except for testosterone, prednisone-metabolite-2 and prednisolone-metabolite-2. The chromatographic conditions used in this study provided clean separation of different natural and synthetic steroids. Approximately 75-85% of the steroids added to plasma and approximately ...
Chung CJ, Grimm AL, Wilson CL, Balasuriya UB, Chung G, Timoney PJ, Bandaranayaka-Mudiyanselage CB, Lee SS, McGuire TC.In an effort to improve a competitive blocking enzyme-linked immunosorbent assay (cELISA) for antibody detection to Equine arteritis virus (EAV), antigen purified by anion-exchange membrane chromatography capsule (AEC) was evaluated. Virus purification by the AEC method was rapid and easily scalable. A comparison was made between virus purified by the AEC method with that obtained by differential centrifugation based on the following: 1) the relative purity and quality of EAV glycoprotein 5 (GP5) containing the epitope defined by monoclonal antibody 17B7, and 2) the relative sensitivity of a c...
Kwok WH, Choi TLS, Leung GNW, Wong ASY, Yue SK, Wan TSM, Ho ENM.The insulin-like peptide relaxin (RLX), an endogenous peptide hormone produced in human for pregnancy and reproduction, is also known to exert a range of physiological and pathological effects. Its use is banned in human sports, horseracing, and equestrian competitions due to its potential performance enhancing effect through vasodilation resulting in the increase of blood and oxygen supplies to muscles. Little is known about the biotransformation and elimination of RLX in horses. This paper describes an administration study of rhRLX-2 and its elimination in horses, and the development of sens...
Ohya T, Kondo T, Yoshikawa Y, Watanabe K, Orino K.In mammal circulation, various ferritin-binding proteins (FBPs) are thought to be involved in the clearance of circulating ferritin after complex formation with it. However, horse FBPs are known to cause inhibitory effects on ferritin immunoassay due to the concealment of the ferritin molecule to anti-ferritin antibodies used in the ferritin immunoassay. These inhibitory effects are eliminated by heat treatment of horse serum at 75°C for 15 min. The inhibitory effects on ferritin immunoassay in the sera of ten foal sera (5 females and 5 males) from 1 to 18 months were detected during all peri...
McDonald J, Gall R, Wiedenbach P, Bass VD, DeLeon B, Brockus C, Stobert D, Wie S, Prange CA, Yang JM.We investigated the use of particle concentration fluorescence immunoassay (PCFIA) as a technique for drug detection in racing horses. The test was constructed from an antiserum to a carboxyfentanyl-BSA conjugate and carboxyfentanyl linked to b-Phycoerythrin. Using these reagents and a PCFIA apparatus levels of fentanyl as low as 0.1 ng/ml could be detected by the assay. In addition, cross-reactivity studies on this assay showed that the anti-serum cross-reacted well with carfentanil, sufentanil and the methylated analogs of fentanyl. We therefore evaluated the ability of these agents to produ...
Hohenhaus MU.A rapid progesterone assay for cow's milk was checked as to whether it was applicable to mares' blood plasma. The "Hygia Progesterone-Test" is an on-farm test which serves for qualitative analysis. It is generally unusable for mares' plasma but sufficiently precise only in cases of larger or smaller progesterone levels. In cases of moderate amounts of progesterone the test is imprecise. The test can be carried out quickly and easily, but the preparation of blood samples takes more time than preparation of milk samples. The test can be recommended for usage in veterinary practice only, but not ...
Niwa H, Anzai T, Hobo S.Contagious equine metritis (CEM) is a highly contagious bacterial venereal disease of horses caused by Taylorella equigenitalis. CEM-PCR is a semi-nested PCR method for detecting this bacterium. Although this technique is regarded as a sensitive diagnostic method for CEM, there are risks of it generating false positive and false negative results. In this study, we constructed a recombinant plasmid (CEM-POS) as reaction control to assure adequate PCR reaction and prevent false positive results caused by contamination of the reaction control in routine CEM-PCR examinations. CEM-POS was construct...
Sukow WW, Bailey J.The binding isotherms for Triton X-100 binding to equine and rabbit serum albumin were determined by equilibrium dialysis at 16 degrees C in pH 7.0, I = 0.05 phosphate buffer. Presented in a Scatchard plot, the binding isotherms are a straight line, indicating thermodynamically independent and identical binding sites. In this model equine serum albumin is characterized as having 11 such sites with an equilibrium constant of 6.0 x 10(3) M-1. Similarly, rabbit serum albumin is characterized as having 9 such sites with an equilibrium constant of 8.0 x 10(3) M-1.
Haywood PE, Chalmers P.The chromatographic and spectroscopic properties of several unusual substances which have been detected in the "alkaloidal" chloroform extract from racehorse urine and saliva samples are reported. Some of these substances have been identified by combined gas chromatography-mass spectrometry and the source of the substance is stated where this is known. Other substances whose identity is not known have been detected and their mass spectra show characteristic amine fragments. The occurrence of these unidentified substances is more frequent in aged urine samples and it would therefore appear that...
Aureli G, Lauria A.The results of a study on interstitial cells of the horse gonads from foetal life to puberty are reported. The morphological (also ultrastructural) histochemical, histophysical and histoenzymological findings both in the organ and in monolayer cultures, clarify the problem of the ontogenesis of these cells showing that: --foetal interstitial cells give origin to "xanthochrome" cells; --"xanthochrome" cells in the prepuberal gonad are continuously renewed; --the same type of cells which in th prepuberal period undergo lipochromic degeneration, differentiate at puberty into Leydig cells in the t...
Beech J, Fletcher JE, Erwin K, Lindborg SR.To determine sensitivity of equine skeletal muscle to tetrodotoxin and compare that with sensitivity of murine and human skeletal muscles. Methods: Semimembranosus, vastus lateralis, triceps brachii, and masseter muscle specimens from 22 euthanatized horses, vastus lateralis muscle biopsy specimens from 25 clinically normal humans, and diaphragmatic muscle specimens from 6 mice. Methods: Electrically elicited twitch responses were measured in muscle specimens incubated in medium alone and with tetrodotoxin (100 nM, 400 nM, 1.6 microM for equine specimens and 100 nM, 200 nM, 400 nM, 800 nM, 1.6...
Schmidt P, Meyer H, Hübert P, Hafner A, Andiel E, Grabner A, Dahme E.The detection of equine herpesvirus type 1 (EHV-1) in infected cell cultures, and in tissues taken at necropsy, by the in-situ hybridization technique is described. A 4.9 kb Bam HI fragment of EHV-1 vaccine strain RacH was used as a probe after labelling with [alpha-32P] thymidine 5'-triphosphate ([32P]TTP) or digoxigenin-deoxyuridine 5'-triphosphate (dUTP). Both probes specifically detected EHV-1 DNA in either cytospin or paraffin wax-embedded preparations of infected cells. The digoxigenin-labelled probe was further used to examine tissue sections of equine fetuses which had been aborted due...
Xu X, Murphy LA.The presence of insecticides like pyrethrins and synthetic pyrethroids, combined with the synergist piperonyl butoxide, in animal feeds can pose a risk to both animal and human health by contaminating the food chain. In this study, a simple and fast method was developed for the simultaneous determination of these compounds in contaminated animal feeds using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample preparation was carried out using a QuEChERS-based approach, and the method was validated with acceptable accuracy ranging from 84 to 115% and precision below 10%. The limit ...
Black WD, Hartley CA, Ficorilli NP, Studdert MJ.Equine rhinitis B virus (ERBV), genus Erbovirus, is most closely related to the Cardiovirus genus in the family Picornaviridae. The structural proteins (VP1-4) of erboviruses are not well described, but are predicted by sequence to be 35, 29, 26 and 7 kDa. Methods for the purification of cardioviruses (polyethylene glycol, trypsin treatment) were used to characterise the structural proteins of ERBV1. Only one of the virus proteins detected was an expected molecular mass, and this 26 kDa protein was identified as VP3 by N-terminal amino acid sequencing. N-terminal sequencing of the 56 and a 29 ...
Craig AM, Blythe LL, Appell LH, Slizeski ML.Dimethyl sulfoxide (DMSO) had been postulated to be a 'masking agent' when used concurrently with therapeutic or prohibited drugs in racing animals. Eight drugs (flunixin, furosemide, caffeine, apomorphine, phenylbutazone, lidocaine, cocaine, and acepromazine maleate) were administered to six horses singly and with concurrent intravenous DMSO. Urine samples were analyzed for the presence of the drugs and/or their metabolites by thin layer chromatography. Direct comparison of thin layer chromatograms of extracts of positive urine samples with and without DMSO verified that DMSO did not interfer...
Weiser MG.Using a single channel electronic cell counter and attached particle size analyzer, leukocyte size distribution histograms were determined on canine, feline, bovine, and equine blood diluted with chloride-based diluent and treated with a conventional stromatolysin. Histograms were usually unimodal, but a few were bimodal. Mean values for mean lysed leukocyte particle volume were 49.2, 51.1, 55.4, and 65.0 fl for canine, feline, equine, and bovine blood, respectively. From inspection of histograms, a lower threshold of 30 fl referenced to latex spheres was interpreted to be appropriate for coun...
Budras KD, Schiel C, Mülling CK, Patan B.The preparation of hard tissues such as the equine hoof horn for electron microscopic examination is very difficult. In particular the penetration of fixatives and chemicals used during fixation and embedding is a problem. The objective of this study was to find and implement an alternative method enabling the preparation of high-quality thin sections of hoof horn and other hard tissue, which maintains the hard tissue ultrastructure and can be used for immuno-labeling. Compared to commonly used fixation and embedding techniques, the preparation of thin sections from untreated material method s...
