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.
Pastor J, Cuenca R, Velarde R, Marco I, Viñas L, Lavín S.A semiautomatic electronic blood cell counter (Sysmex F-800) was evaluated with equine blood, according to the protocol of the International Committee for Standardization in Haematology (ICSH, 1984). The precision and overall reproducibility were acceptable for all the parameters studied except for the platelet count, in which a coefficient of variation of 18.8% and 21.7% was obtained for within and between batch precision and 26.76% for overall reproducibility. Carry-over for the haematocrit value and platelet count was unsatisfactory, thus the use of a blank diluent sample between different ...
Parillo F, Stradaioli G, Supplizi AV, Monaci M.This study was undertaken to determine the lectin affinity of the extratesticular rete testis and ductuli efferentes epithelial cells in adult and prepubertal horses, using ten different lectin horseradish peroxidase conjugates: Con-A, LCA, WGA, GSA-II, SBA, PNA, RCA-I, DBA, UEA-I, and LTA. In some cases, treatments with sialidase and KOH preceded the lectin staining. In sexually mature and immature horses the results showed the presence of different kinds of sialoglycoconjugates with the terminal sialic acid linked to D-GalNAc and beta-D-Gal residues in the rete testis. In the apical surface ...
Eggleston-Stott ML, Delvalle A, Dileanis S, Wictum E, Bowling AT.The equine dinucleotide microsatellite HMS7 is part of a microsatellite panel utilized in a parentage verification programme at the Veterinary Genetics Laboratory (Davis, California, USA). Apparent non-Mendelian inheritance was noted when a Quarter Horse mare was excluded as the parent of two offspring based on analysis of the HMS7 locus. The mare's DNA type qualified her as a parent of the offspring at an additional 20 microsatellite loci. The three animals appeared homozygous for HMS7 with each possessing an allele different from that of the other two animals. Polymerase chain reaction prime...
Chou CC, Chen CL, Asbury AC, Webb AI, Vickroy TW.To develop an ELISA that is sensitive and suitable for measurement of immunoreactive acepromazine (ACP) in horse serum and urine and to determine the acute effects of exercise on immunoreactive ACP values in Thoroughbreds. Methods: 12 healthy Thoroughbreds (5 mares, 5 geldings, 2 stallions), aged 2 to 8 years. Methods: A commercially available antibody and a horseradish peroxidase-conjugated oxime derivative of immunoreactive ACP were used to develop a one-step ELISA. Horses were used in a crossover design study to evaluate possible effects of treadmill exercise on serum and urine ACP concentr...
Topper MJ, Prasse KW.To adapt manual human chromogenic assays for coagulation factors VII (F.VII), VIII:coagulant (F.VIII:C), IX (F.IX), and X (F.X), and C1-esterase inhibitor (C1-INH) for use with an automated analyzer, and to measure the activity of these proteins in horses. Methods: 10 healthy horses were used to determine ranges for the assays. Pooled plasma for standards was collected from an additional 20 healthy horses. Methods: A computer-assisted analyzer was programmed from the manual method for commercially available human F.VII, F.VIII:C, F.IX, F.X, and C1-INH chromogenic assay kits. Standards were pre...
Liang FT, Granstrom DE, Zhao XM, Timoney JF.Sarcocystis neurona is the etiologic agent of equine protozoal myeloencephalitis (EPM). Based on an analysis of 25,000 equine serum and cerebrospinal fluid (CSF) samples, including samples from horses with neurologic signs typical of EPM or with histologically or parasitologically confirmed EPM, four major immunoblot band patterns have been identified. Twenty-three serum and CSF samples representing each of the four immunoblot patterns were selected from 220 samples from horses with neurologic signs resembling EPM and examined for inhibitory effects on the infectivity of S. neurona by an in vi...
Sternberg S.Swab samples were collected from the oral cavity of 174 horses in 10 farms and cultured selectively for Actinobacillus equuli. A. equuli could be isolated from 37% of all samples, varying between 12 and 88% in the different farms. Eight horses were sampled repeatedly for several days, with a variation in isolation frequency between 50 and 88%. Isolates were compared by restriction enzyme digestion and Pulsed-Field Gel Electrophoresis. A high degree of strain variability was found within each horse population as well as some variability over time between strains isolated from the same horse.
Dyke TM, Sams RA.The objective of this study was to determine the urinary excretion of methylxanthines in horses following ingestion of chocolate over eight days. The study was performed in response to gas chromatography-mass spectrometry (GC-MS) confirmation of the presence of caffeine in a positive urine test in a racehorse. The trainer of the horse alleged that he often administered chocolate-coated peanuts as treats to his horses, and he believed that the ingestion of chocolate was responsible for the positive urine test. The urinary excretion of theobromine and caffeine after the ingestion of chocolate-co...
Bailey SR, Elliott J.Cumulative concentration response curves to 5-hydroxytryptamine (5-HT; 10(-10)-10(-4) mol/l) were constructed using isolated rings of equine digital, facial, tail and coronary arteries (endothelium intact). 5-HT was 17.7 and 41 times more potent as a vasoconstrictor of digital arteries than facial and tail arteries respectively. Removal of the endothelium increased the vasoconstrictor potency of 5-HT in the facial artery by 3.7-fold (P<0.05) but did not alter the sensitivity of digital arteries to 5-HT. Coronary arteries failed to contract to 5-HT. Coronary arteries pre-contracted with U440...
Haouz A, Glandieres JM, Zentz C, Pin S, Ramstein J, Tauc P, Brochon JC, Alpert B.The effects of the solvent conditions (buffer pH 9, 8, or 7 or buffer pH 6.5 alone or mixed with 3.2% ethanol or 6.2% formamide) on the protein dynamics of horse apomyoglobin were investigated through tryptophan fluorescence quenching, spectra, and decay properties. Raising the pH (which induces discontinuous protein conformation changes) increases the structural fluctuations inside the hydrophobic A, G, and H helix core. Mixed solutions containing either 3.2% ethanol or 6.2% formamide (which redistribute water molecules on the protein surface) produce protein dynamics changes in the vicinity ...
Brugmans F, Venner M, Menzel D, Mischke R.The purpose of this study was to investigate the usefulness of two heat-precipitation techniques (Schalm- and Millar-method) as screening tests to measure plasma fibrinogen concentration in horses. Based on the measurement of samples from 108 different horses, the coefficient of correlation (CC) for the relationship between the results with the Schalm- and with the reference-method (Jacobsson) were much lower (r = 0.78) than between the Millar- and Jacobsson-method (r = 0.94). Furthermore the Schalm-method was less precise as reflected by the greater coefficient of variation (CV, within-run pr...
Dyke TM, Sams RA, Hinchcliff KW.To measure renal clearance of antipyrine and urinary excretion of antipyrine (AP) metabolites in horses by use of validated high-performance liquid chromatography (HPLC) methods. Methods: 8 Standardbred mares. Methods: HPLC methods for measurement of AP in equine plasma and AP and its metabolites in equine urine were validated. Antipyrine (20 mg/kg of body weight) was administered i.v., and blood samples and urine specimens were collected over 24 hours. Results: Median plasma clearance of AP in horses was 6.2 ml/min/kg, of which < 2% could be attributed to renal clearance. Urinary excretion...
Stanley SM, Kent S, Rodgers JP.In this study the equine metabolism of fluoxymesterone (9alpha-fluoro-11beta-17beta-dihydroxy-17alpha-meth ylandrost-4-ene-3-one) given orally has been investigated. The parent material was not detected, but two major 16-hydroxy metabolites which corresponded to a mono- and a di-hydroxylation product were evident. One of the hydroxylation positions was identified as C-16. Phase II metabolism in the form of glucuronide formation was also common. These steroids will provide target compounds for confirming abuse of this drug in the horse.
Delbeke FT, Baert K, De Backer P.Urinary and plasma concentrations of the nonsteroidal anti-inflammatory drug tiaprofenic acid were determined following oral and intramuscular administration of a dose of 1 g to five fasted horses. Quantitation was performed by high-performance liquid chromatography (HPLC). The limit of quantitation (LOQ) was 0.1 microg/ml and 0.5 microg/ml in 2 ml plasma and 1 ml urine, respectively. Assay precision and extraction recovery were between acceptable values. Tiaprofenic acid pharmacokinetics were described by non-compartment analysis of the data. Absorption was faster after oral administration as...
van den Hoogen BM, van Weeren PR, Lopes-Cardozo M, van Golde LM, Barneveld A, van de Lest CH.The amount of uronic acid residues in samples containing glycosaminoglycans or pectin is an important parameter in the quantitative and structural analysis of these complex carbohydrates. This paper describes a method to determine the content of uronic acids in biological samples, using conventional polystyrene microtiter plates and microtiter plate-reading equipment with standard interference filters (i.e., 540 or 492 nm). This assay is a modification of a commonly used procedure, viz. hydrolysis of uronic acid containing carbohydrate polymers in 80% sulfuric acid containing tetraborate ions ...
Goudet G, Leclercq L, Bézard J, Duchamp G, Guillaume D, Palmer E.This study reports the follicular growth and oocyte competence for in vitro maturation and fertilization under the influence of circulating eCG. Three to 7 successive ultrasound-guided follicular punctures were performed on 4 pregnant mares from Day 23 until Day 75 of pregnancy and on 5 control mares whose embryonic vesicle was crushed on Day 22. All follicles larger than 5 mm were punctured 24 h after the largest follicle reached 18 mm. Expanded cumulus oocyte complexes (COCs) were stained at recovery to analyze the nuclear stage. Compact COCs were cultured in vitro for 46 h and either staine...
Meana A, Luzon M, Corchero J, Gómez-Bautista M.Three coprological methods were tested to establish the reliability of in vivo diagnosis of Anoplocephala perfoliata. A total of 107 faecal samples were analyzed, and the presence of tapeworms were confirmed postmortem in 24 animals with burdens that ranged from 1 to 248 worms; most of them (71%) with less than 100 parasites. Best results were obtained with a combination of two sedimentation/flotation methods, detecting only half the parasitized animals (54% sensitivity). No relationship could be established between tapeworm burden and egg detection, but results indicate that coprological meth...
Brooks DE, Andrew SE, Dillavou CL, Ellis G, Kubilis PS.To evaluate in vitro susceptibility to topical antifungal medications, as measured by minimum inhibitory concentration (MIC) and 50% inhibitory concentration (IC50%), of fungal isolates from horses with ulcerative keratomycosis in Florida; to compare results with those of other studies to identify differences in susceptibility patterns among fungi isolated from horses in different geographic regions; and to note indications of fungal resistance to drugs tested in other studies. Methods: Corneal fungal cultures from client-owned horses from Florida with ulcerative keratomycosis (n = 22). Method...
Koepf EK, Hewitt J, Vo H, Macgillivray RT, Burtnick LD.We have generated and characterized the cDNA from equine smooth muscle that encodes gelsolin, an actin-modulating protein. Overlapping cDNA clones synthesized by the reverse transcriptase/polymerase chain reaction and clones isolated from a horse genomic library provided the complete primary structure for the intracellular isoform of gelsolin, while cDNA complemented with protein sequence data produced the full-length primary transcript of the gelsolin isoform found circulating in equine plasma. The deduced amino acid sequences of the intracellular and secreted versions of equine gelsolin infe...
Sahagun-Ruiz A, Waghela SD, Holman PJ, Chieves LP, Wagner GG.A DNA probe from Babesia caballi (Bc1) was selected by antibody screening of a genomic library. The Bc1 probe hybridized specifically to B. caballi genomic DNA. A polymerase-chain-reaction-based assay for B. caballi DNA was developed from primers deduced from the probe nucleotide sequence. An amplified product of 1.6 kb was detected from as little as 500 fg B. caballi template DNA. Sensitivity increased 1000-fold when the biotin-labeled Bc1 probe was hybridized to the amplicons in a Southern blot.
Carter RA, Ericsson SA, Corn CD, Weyerts PR, Dart MG, Escue SG, Mesta J.The objective of this study was to determine if spermatozoal reduction of the dyes methylene green to colorless and resazurin to pink or colorless was associated with the fertility potential of an equine semen sample. Fifty samples from 38 stallions were evaluated for the number of spermatozoa per milliliter and number of motile sperm per milliliter. Methylene green (20 micrograms/mL of semen) or resazurin (85 micrograms/mL of semen) was added to 3-mL aliquots of semen. Semen samples were identified as having low fertility potential (< 200 x 10(6) total cells/mL and or = 200 x 10(6) total ...
Larsen M, Nansen P, Grønvold J, Wolstrup J, Henriksen SA.The potential of using fungi to prevent nematodosis caused by parasites with free-living larval stages is well documented today. In this respect Duddingtonia flagrans, a net-trapping, nematode-destroying fungus, appears to be the most promising candidate. Laboratory experiments and in-vivo studies, where fungal spores have survived passage through the gastro-intestinal tract of cattle and horses, plus field studies with cattle, horses and pigs, demonstrate significant reduction in the number of infective larvae that develop in the faecal environment. In field trials this reduction subsequently...
Zhang XY, Zhu FX, Olszewski MA, Robinson NE.The beta 2-agonists currently used as bronchodilators are racemic mixtures of R- and S-enantiomers. In the present study, we examined the effects of enantiomers of the beta 2-agonists albuterol and formoterol on acetylcholine (ACh) release from equine trachealis parasympathetic nerves. ACh release was evoked by electrical field stimulation (20 V, 0.5 ms, 0.5 Hz) and measured by high-performance liquid chromatography coupled with electrochemical detection. We also tested the effects of enantiomers of albuterol and formoterol on equine tracheal smooth muscle (TSM) contraction in response to exog...
Magdaleno L, Gasset M, Varea J, Schambony AM, Urbanke C, Raida M, Töpfer-Petersen E, Calvete JJ.HSP-3 is a member of the cysteine-rich secretory protein (CRISP) family from stallion seminal plasma. We report a large-scale purification protocol for native HSP-3. This protein is a non-glycosylated polypeptide chain with a pI of 8-9 and an isotope-averaged molecular mass of 24987 +/- 3 Da. The molecular mass of HSP-3, determined by equilibrium sedimentation, is 26 kDa, showing that the protein exists in solution as a monomer. The concentration of HSP-3 in the seminal plasma of different stallions ranged from 0.3 to 1.3 mg/ml. On average, 0.9-9 million HSP-3 molecules/cell coat the postacros...
Holland PS, Brumbaugh GW, Ruoff WW, Brown SA.Plasma pharmacokinetics of ranitidine HCl were investigated after intravenous (i.v.) and oral (p.o.) administration of drug to six healthy foals. Twelve- to sixteen-week-old foals received 2.2 mg ranitidine/kg i.v. and 4.4 mg ranitidine/kg p.o. Concentrations of ranitidine were determined using normal phase high performance liquid chromatography. Plasma concentrations of ranitidine HCl declined from a mean of 3266 ng/mL at 5 min to 11 ng/mL at 720 min after administration. The profile of the plot of concentrations of ranitidine HCl vs. time was best described by a two-exponent equation for two...
Mizuguchi M, Nara M, Ke Y, Kawano K, Hiraoki T, Nitta K.Interactions between Ca2+ and the Asp side chains in the Ca2+-binding site of equine lysozyme were investigated by Fourier-transform infrared (FT-IR) spectroscopy. In the spectrum of equine lysozyme, the intensities of the bands at about 1595 cm-1 and 1578 cm-1 in the region of the COO antisymmetric stretches increased upon Ca2+ binding. In the region of the COO- symmetric stretches, the loss of intensity at about 1388 cm-1 and gains of intensities at about 1423 cm-1 and 1403 cm-1 were observed due to Ca2+ binding to equine lysozyme. The spectral changes for equine lysozyme indicate that the C...
Pang H, Kong XG, Sentsui H, Kono Y, Sugiura T, Hasegawa A, Akashi H.Six strains of equine infectious anemia virus (EIAV) were recovered from febrile and non-febrile stages of a horse experimentally infected with the P337-V70 strain given once to a horse. The env gp90 genes of the isolates, the P337-V70 and P337-V26, avirulent virus derived from the P337-V70 strain, were sequenced. A comparison of the gp90 gene sequences revealed that amino acid variations among the viruses tested showed as high as 8.2 to 11.5%. In addition, the comparison also indicated that the isolates that recovered from the non-febrile stage were contained in nucleotide insertions in the p...
Aguilera R, Becchi M, Mateus L, Popot MA, Bonnaire Y, Casabianca H, Hatton CK.A gas chromatography-combustion-isotope ratio mass spectrometry method for confirmation of hydrocortisone abuse in horseracing and equine sports is proposed. Urinary hydrocortisone was converted to a bismethylenedioxy derivative which presents good gas chromatographic properties and brings an extra carbon contribution of only two carbon atoms. Synthetic hydrocortisone has a different 13C abundance from that of natural urinary horse hydrocortisone and the difference is significant, therefore exogenous and endogenous hydrocortisone can be distinguished.
Shibany KA, Pratt SL, Aldurdunji M, Totemeyer S, Paine SW.Horses are exposed to various kinds of medication, however, there are limited determinations of plasma clearance (CL) for the drugs used due to the high cost of equine studies.Many of the CL values generated come from the equine sports industry for determining drug plasma screening limits in the control of medications at the time of competition.The kinetics of omeprazole metabolism were investigated in freshly isolated and cryopreserved equine hepatocytes and hepatic microsomes ( = 3 horses).The V, K and intrinsic clearance (CL) of omeprazole were determined via the substrate depletion me...
Marsh MV, Caldwell J, Hutt AJ, Smith RL, Horner MW, Houghton E, Moss MS.The metabolism of benzoic acid has been examined in the horse, using 14C- and deuterium-labelled compounds. Chromatographic analysis of the urine showed the presence of hippuric acid, benzoyl glucuronide and benzoic acid and a discrete band which accounted for 2% of the dose administered. This material was isolated by solvent extraction and HPLC and, following treatment with diazomethane, examined by GC/MS. The major component of this fraction was 3-hydroxy-3-phenylpropionic acid methyl ester, which was accompanied by very much smaller amounts of cinnamic acid methyl ester and acetophenone. Th...
Singha H, Goyal SK, Malik P, Singh RK.Thermostabilizing effect of heavy water (D2O) or deuterium oxide has been demonstrated previously on several enzymes and vaccines like oral poliovirus vaccine and influenza virus vaccine. In view of the above observations, effect of heavy water on in situ thermostabilization of recombinant p26 protein on enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of equine infectious anemia virus (EIAV) infection was investigated in the present study. The carbonate-bicarbonate coating buffer was prepared in 60% and 80% D2O for coating the p26 protein in 96-well ELISA plate and thermal stabilit...
O'Donovan LH, McMonagle EL, Taylor S, Argyle DJ, Nicolson L.Interleukin 18 (IL-18) is a cytokine capable of induction of IFNgamma, granulocyte monocyte-colony stimulating factor (GM-CSF), TNFalpha and IL-1 in immunocompetent cells. Equine and feline plasmid vectors expressing pro-IL-18, mature IL-18 and IL-18 fused to a synthetic signal sequence from human IL-1beta receptor antagonist protein (ILRAP), ILRAP-IL-18, have been generated. In vitro protein expression of these constructs was compared by Western blot analysis. These data demonstrated that ILRAP-IL-18 protein was secreted readily from transfected chinese hamster ovary (CHO) cells. A simple bio...
Ward RJ, Ramsey M, Dickson DP, Hunt C, Douglas T, Mann S, Aquad F, Peters TJ, Crichton RR.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...
Prickett TCR , Inder WJ, Evans MJ, Donald RA.The acute-phase cytokine interleukin-1 (IL-1) is known to activate the hypothalamic pituitary adrenal axis, primarily via corticotropin releasing hormone (CRH). The aim of this study was to determine whether IL-1beta could directly stimulate ACTH secretion from perifused equine anterior pituitary cells, and whether CRH pre-incubation affected corticotroph responsiveness. Isolated equine anterior pituitary cells were pre-incubated with media containing 10 nM CRH or vehicle for 20 hours before being loaded onto columns and perifused with 0.02 nM CRH and 100 nM cortisol. Columns were given a 5-mi...
Bozorgmanesh R, Sutton-Burges JW, Tablin F.Equine whole blood collection and storage methods have been evaluated to assess red blood cell viability; however, platelet (PLT) viability has not been comprehensively assessed. Objective: The purpose of the study was to compare viability of PLTs collected in whole blood into 2 different anticoagulants. Methods: Whole blood from 6 healthy adult Thoroughbred horses was collected into citrate-phosphate-dextrose-adenine (CPDA) or acid-citrate-dextrose (ACD). Platelet count, pH, and concentrations of glucose, lactate, carbon dioxide, oxygen, bicarbonate, sodium, potassium, and chloride were measu...
Hochi S, Fujimoto T, Choi YH, Braun J, Oguri N.Immature equine oocytes were frozen-thawed with ethylene glycol (EG), 1,2-propanediol (PD) or glycerol (GL) in PBS and cultured to assess the rate of in vitro maturation (Experiment 1). Compact-cumulus oocyte complexes were collected from slaughterhouse ovaries and equilibrated for 10 min in the freezing medium containing 10% (V/V) cryoprotectant and 0.1 M sucrose. The 0.25-ml straws, loaded with 10 to 30 oocytes, were seeded at -6 degrees C and cooled to -35 degrees C at 0.3 degrees C/min before being plunged into liquid nitrogen. The straws were thawed rapidly in a 37 degrees C waterbath for...
Bolann BJ, Ulvik RJ.The redox interaction between O2.- and ferritin cannot solely be regarded as as a Fe(II) release reaction. We demonstrate that native copper bound to horse spleen ferritin and apoferritin, stimulated the decay of O2.- in a catalytic reaction. Copper was determined by atomic absorption spectrophotometry. Decay of O2.- was monitored spectrophotometrically as the decrease in (A250-A360) at pH 9.5. The catalytic effect was linearly related to the copper content of the protein. Ferritin copper was less efficient than equimolar CuCl2, and iron-poor ferritin was more efficient than iron-rich ferritin...
Gim JA, Kim HS.MicroRNAs (miRNAs) are single-stranded, small RNAs (21-23 nucleotides) that function in gene silencing and translational inhibition via the RNA interference mechanism. Most miRNAs originate from host genomic regions, such as intergenic regions, introns, exons, and transposable elements (TEs). Here, we focused on the palindromic structure of medium reiteration frequencies (MERs), which are similar to precursor miRNAs. Five MER consensus sequences (MER5A1, MER53, MER81, MER91C, and MER117) were matched with paralogous transcripts predicted to be precursor miRNAs in the horse genome (equCab2) and...
Lu Z, Kasaragod DK, Matcher SJ.We present a phase fluctuation calibration method for polarization-sensitive swept-source optical coherence tomography (PS-SS-OCT) using continuous polarization modulation. The method uses a low-voltage broadband polarization modulator driven by a synchronized sinusoidal burst waveform rather than an asynchronous waveform, together with the removal of the global phases of the measured Jones matrices by the use of matrix normalization. This makes it possible to average the measured Jones matrices to remove the artifact due to the speckle noise of the signal in the sample without introducing aux...
Evans MJ, Marshall AG, Kitson NE, Summers K, Donald RA.The multifactorial control of ACTH is well established. We wished to establish and characterize an in-vitro perifusion system, using equine anterior pituitary cells and physiological concentrations of secretagogues, to investigate factors which affect the dynamics of ACTH secretion. Anterior pituitary tissue was divided for dispersion into cells with collagenase, trypsin or dispase, or by mechanical dispersion. After dispersal followed by 18-h incubation, cells were perifused and the ACTH response to 10-min pulses of arginine vasopressin (AVP; 100 nmol/l), corticotrophin-releasing hormone (CRH...
Bougrine SI, Fihri OF, Fehri MM.A Western immunoblotting procedure has been developed for the detection of African horse sickness virus (AHSV) protein-specific antibody responses. This assay readily identifies antibodies specific for at least 4 distinct, AHSV proteins, including VP5, NS1, NS2 and NS3/NS3a. By using the AHSV non-structural proteins as 'markers', the Western blotting procedure could be employed to provide a reliable means of discriminating between animals vaccinated with a purified, inactivated AHSV vaccine and those either naturally infected or vaccinated with a live, attenuated AHSV vaccine.
Bramucci M, Miano A, Quassinti L, Maccari E, Murri O, Amici D.This study was conducted to determine the presence of the angiotensin converting enzyme in commercial sera used in cell culture medium. The aim of the research was to bring the presence of proteinases (angiotensin converting enzyme) to cell culture users' knowledge and to give some data for solving problems about the development of peptides as useful drugs. The enzymes, purified from foetal bovine, adult bovine, foetal equine, adult equine, and human sera, showed molecular weights of about 170 kDa. Captopril and lisinopril inhibited enzyme activities at nanomolar concentrations. The enzymes we...
Bonfini B, Semproni G, Savini G.A horse erythrocyte culture technique, partly modifying that originally developed by Holman, was used to detect the presence of Theileria equi strains in 12 horse and 2 mule blood samples. The animals were placed into four groups on the basis of their case history and laboratory test results: the mules and two horses were considered as infected and included in the 'recent infection' group, four horses with a history of past infection were included in the 'past infection' group and four animals subjected to anti-theileria treatment formed the 'treated animals' group. The final group consisted o...
Brink P, DeGraves F, Ravis WR, Johansen D, Campbell JD, Duran SH.To determine intravascular and intrasynovial pharmacokinetics of the R and S enantiomers of ketoprofen after i.v. and i.m. administration to horses. Methods: 6 healthy adult mares. Methods: Horses were weighed and ketoprofen (2.2 mg/kg of body weight) was administered i.v. Blood and synovial fluid samples were obtained and analyzed for concentrations of the R and S enantiomers by means of a modified reverse-phase stereospecific high-pressure liquid chromatographic method. Three weeks later, the procedure was repeated, except that ketoprofen was given IM. Protein binding of ketoprofen enantiome...
Archambault D, St-Louis MC, Martin S.The genome of equine arteritis virus (EAV) produces a 3' coterminal-nested set of six subgenomic (sg) viral RNAs during virus replication cycle, and each set possesses a common leader sequence of 206 nucleotides (nt) in length derived from the 5' end of the viral genome. Given the presence of the leader region within both genomic and sg mRNAs, it is likely to contain cis-acting signals that may interact with cellular or viral proteins for RNA synthesis. Gel mobility shift assays indicated that proteins in Vero cell cytoplasmic extracts formed complexes with the positive (+) and negative (-) st...
Pedersen CE, Eddy GA.Polyacrylamide gel electrophoretic examination of viruses selected from the Venezuelan equine encephalomyelitis (VEE) complex revealed distinct strain to strain differences in profiles of the two virion envelope proteins. The core protein was identical in all viruses tested. We detected five electrophoretic patterns into which the virus strains could be classified and these were designated alpha (alpha), beta (beta), gamma (gamma), delta (delta), and episolon (episolon). Isolates representing variant E of subtype I exhibited a profile characterized by only one apparent envelope band. The epizo...
Kent JE, Blackmore DJ.This paper describes the quantitative measurement of IgG concentrations in equine blood/serum by turbidimetric analysis and the qualitative assessment using latex agglutination. The concentration of IgG in foal serum measured by turbidimetry correlated well with the results obtained by radial immunodiffusion (r = 0.91) and the gamma-globulins estimated from the electrophoresis of serum on cellulose acetate (r = 0.92). The method, using antibody-coated latex beads, to detect foals with serum IgG concentrations of less than 4 g/litre (whole blood less than 2 g/litre) proved to be accurate in 96 ...
Di Filippo PA, Vieira V, Rondon DA, Quirino CR.The aim of the study was to determine if dental correction changes fecal fiber length (FFL) in horses that had never been subjected to dental treatments. Thirty adult horses with mild to moderate dental abnormalities were selected. Dental disorders noted included sharp enamel points, focal overgrowths, shear mouth, step mouth, wave mouth, accentuated transverse ridges, and diastema. The horses were confined to individual stalls and fed Tifton hay (Cynodon spp.) 1.5% bwt/d, divided equally between two feedings. Fecal samples were taken for FFL analysis before (Day 0) and after (Day 15) dental c...
Strizki JM, Repik PM.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...
Merlo B, Iacono E, Bucci D, Spinaci M, Galeati G, Mari G.In vitro embryo production in the horse is still not as efficient as in other species. Oxidative stress negatively affects oocyte and embryo culture. To attenuate/minimize the oxidative stress, antioxidants such as low-molecular thiol compounds can be added to culture media. Beta-mercaptoethanol (BME) has been shown to improve maturation and embryo development in different species. The aim of this study was to investigate whether the addition to maturation medium of BME at common (0.1 mM) and high (0.7 mM) concentration could improve oocyte maturation also in the horse. Equine oocytes recove...
Guan F, You Y, Li X, Robinson MA.α-Cobratoxin (CTX) is a large peptide (71 amino acids) with strong analgesic effect and may be misused in sports such as horse racing. To prevent such misuse, a sensitive method is required for detection and confirmation of the toxin in equine samples. CTX was extracted from equine plasma using an optimized mixed-mode solid-phase extraction (SPE) procedure. Extracted CTX was reduced with dithiothreitol and alkylated with iodoacetamide, and then was digested by trypsin at 56°C for 30min. The digest was analysed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and trypt...
Waraksa E, Woźniak MK, Kłodzińska E, Wrzesień R, Bobrowska-Korczak B, Namieśnik J.Ibuprofen is widely used in human and veterinary medicine for the treatment of chronic pain as well as rheumatic and musculoskeletal disorders. However, the analgesic and anti-inflammatory properties of Ibuprofen have contributed to frequent drug abuse in equestrian sports. A sensitive and rapid gas chromatography with tandem mass spectrometry based method with a simple liquid-liquid extraction and derivatization requiring 200 μL volume of sample and 2 mL of extraction solvent for the simultaneous determination of ibuprofen and its metabolites was developed. The proposed procedure was optim...
Morris DD, Moore JN.Equine peritoneal macrophages were isolated and cultured in vitro to assess their ability to produce thromboxane (TxA2) and prostacyclin (PGI2) in response to endotoxin. Peritoneal macrophages (2.5 x 10(6)/ml) were incubated in tissue culture media, containing 1) no additive (nonstimulated control), 2) endotoxin (0.5 to 100 ng/ml) or 3) the calcium ionophore, A23187 (0.95 microM) for two and six h. Concentrations of the stable metabolites of TxA2 and PGI2 thromboxane B2 (TxB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), in the incubation media were determined by radioimmunoassay. Th...
Brinkmeyer-Langford CL, Murphy WJ, Childers CP, Skow LC.The assembled genomic sequence of the horse major histocompatibility complex (MHC) (equine lymphocyte antigen, ELA) is very similar to the homologous human HLA, with the notable exception of a large segmental duplication at the boundary of ELA class I and class III that is absent in HLA. The segmental duplication consists of a ∼ 710 kb region of at least 11 repeated blocks: 10 blocks each contain an MHC class I-like sequence and the helicase domain portion of a BAT1-like sequence, and the remaining unit contains the full-length BAT1 gene. Similar genomic features were found in other Perissod...
Betton JM, Desmadril M, Mitraki A, Yon JM.The kinetics of the unfolding and refolding of horse muscle phosphoglycerate kinase were studied with three different signals: fluorescence emission intensity at 336 nm (excitation at 292 nm), ellipticity at 220 nm, and enzyme activity. The results corroborate the conclusion on the existence of intermediates in the folding pathway obtained from equilibrium studies. Kinetic studies showed at least two phases of refolding, as revealed by fluorescence as well as by circular dichroism measurements. During the fast phase, an intermediate was formed with a fluorescence intensity higher than that of ...
Heo EJ, Lee HS, Jeoung HY, Ko HR, Kweon CH, Ko YJ.A recombinant glycoprotein (R-GP) of vesicular stomatitis New Jersey virus (VSV-NJ) was expressed in insect cells by a baculovirus system. Its utility as a diagnostic antigen in a blocking ELISA was investigated as an alternative to the current native GP extracted from VSV-NJ. With the cut-off value of 73% inhibition, the R-GP ELISA exhibited 99.1% specificity for naive sera from cattle and horses. It did not cross-react with VSV-Indiana (VSV-IN) positive sera and differentiated from foot-and-mouth disease and swine vesicular disease. Taken together, this is the first report that the R-GP has ...
John HA, Marrs J, Laffling AJ.In the autonomic nervous system (ANS) of equine grass sickness (GS) cases, some neurones show abnormal changes while neighbouring neurones are unaffected. To test whether noradrenergic neurones showed variable susceptibility to the GS toxin in culture, clonally-derived populations isolated from the same fetal thoracic sympathetic chain ganglion were challenged with plasma from GS cases previously shown to induce ANS damage when injected into normal horses. During the early stages of exposure to toxic plasma, cells within a clonal population showed variable susceptibility ranging from no obviou...
Lopez I, Estepa JC, Mendoza FJ, Mayer-Valor R, Aguilera-Tejero E.To establish reference values for protein-bound, ionized, and weak-acid complexed fractions of calcium and magnesium in equine serum and determine stability of ionized calcium (iCa) and ionized magnesium (iMg) in serum samples kept under various storage conditions. Methods: 28 clinically normal horses. Methods: Total calcium (tCa) and magnesium (tMg) in equine serum were fractionated by use of a micropartition system that allows separation of protein-bound calcium (pCa) and magnesium (pMg) and ultrafiltrable calcium (microCa) and magnesium (microMg) fractions. Serum concentrations of iCa and i...
