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.
Csellner H, Walker C, Love DN, Whalley JM.An equine herpesvirus 1 (EHV-1) mutant was constructed by inserting a lacZ expression cassette into the intergenic region upstream of gene 62 (glycoprotein L; gL) and downstream of gene 63 (a homologue of the herpes simplex virus transcriptional activator ICP0). The recombinant lacZ62/63-EHV-1 had similar growth kinetics in cell culture to those of the parental wild type (wt) virus, with indistinguishable cytopathic effects and plaque morphology. Reverse transcriptase PCR confirmed that the lacZ insertion did not interfere with transcription of gL and immunoblot analysis indicated there was no...
Vanin AF, Serezhenkov VA, Mikoyan VD, Genkin MV.The parameters of EPR signal from dinitrosyl-iron complexes (DNIC) with bovine serum albumin (BSA), horse hemoglobin (Hb), and apometallothionein (apo-Mt) of horse kidney incorporating one (BSA, Hb) or two thiol-containing ligands (apo-Mt) were compared. The EPR signal from DNIC-BSA was characterized by the rhombic symmetry of g tensor at room temperature of signal recording (ambient temperature) or at 77K in the solution frozen in the presence of glycerol. In freezing of the solution in the absence of glycerin, under the exposure of DNIC-BSA to negatively charged sodium dodecyl sulfate (SDS) ...
Bailey DN.The relative binding of acetaminophen, lidocaine, phenobarbital, procainamide, quinidine, and theophylline to sera of seven mammalian species was studied. Pooled commercial sera from cow, goat, horse, human, pig, rabbit, and sheep were supplemented with 5 and 10 mM concentrations of each drug. For each serum, each drug, and each drug concentration, equilibrium dialysis was performed in duplicate against phosphate buffer (pH 7.4, 0.1 M, 4 degrees C). Percent drug bound to serum was calculated. Phenobarbital demonstrated more than 20% binding to goat, horse, human, and sheep serum at both 5 and ...
Estepa JC, Aguilera-Tejero E, Mayer-Valor R, Almadén Y, Felsenfeld AJ, Rodríguez M.Measurement of parathyroid hormone (PTH) in horses was performed on plasma samples using 2 immunoradiometric assays: a human intact PTH assay and a rat amino-terminal PTH assay. The assays were validated by assessment of their precision, sensitivity and specificity, and also by evaluating PTH changes in the horse in response to variation in blood ionised calcium. Intra- and inter-assay variance, precision and sensitivity were similar for both human and rat assays; however, the rat assay was slightly more precise and sensitive than the human assay. Both assays detected an increase in PTH levels...
Gray AW, Davies ME, Jeffcott LB.We report on preliminary results of a novel in vitro culture system designed to generate equine osteoclasts in large numbers. Osteoclast generation, as determined by the expression of tartrate resistant acid phosphatase (TRAP) and ability to resorb bone, was enhanced in equine bone marrow cultures supplemented with fibroblastic cell (L929) conditioned medium (L929-CM). Bone marrow was collected from a total of 12 horses and ponies and TRAP-positive cells with bone resorbing ability were generated in significant numbers in the last seven. TRAP-positive mononuclear cells appeared after three day...
Okumura M, Tagami M, Fujinaga T.Keratan sulphate (KS) concentration and anticollagen type II antibody levels were measured in the serum and synovial fluid (SF) of clinically normal horses and horses with osteoarthritis (OA). Serum KS in OA was significantly higher than that in normal horses, while no significant difference was found in KS levels of SF between normal and OA. Assays of antibody to collagen type II showed no significant increase in sera and SF of OA. It was suggested that levels of serum KS would be of value in the pathological detection of OA in the joint, although there was no evidence that the measurable aut...
Nagamine N, Nambo Y, Nagata S, Nagaoka K, Tsunoda N, Taniyama H, Tanaka Y, Tohei A, Watanabe G, Taya K.To determine the source of circulating inhibin and estradiol-17beta during the estrous cycle in mares, the cellular localization of the inhibin alpha, betaA, and betaB subunits and aromatase in the ovary was determined by immunohistochemistry. Concentrations of immunoreactive (ir-) inhibin, estradiol-17beta, progesterone, LH, and FSH in peripheral blood were also measured during the estrous cycle in mares. Immunohistochemically, inhibin alpha subunits were localized in the granulosa cells of small and large follicles and in the theca interna cells of large follicles, whereas inhibin betaA and ...
Yasunaga S, Maeda K, Matsumura T, Kai K, Iwata H, Inoue T.Recently, a type-specific ELISA using equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4) glycoprotein Gs (gGs) was developed by Crabb and Studdert [1993]. To investigate the dissemination of EHV-1 and -4 among horses in Japan, we applied their ELISA as suitable for discriminating between EHV-1 and -4 infections serologically. Type-specificity of the ELISA was confirmed by using paired sera of infected horses with either EHV-1 or -4. Application of the ELISA to sera collected before and after the winter season of 1995-1996 from 80 racehorses revealed that 30 horses showed significant antibody...
Krüger B, Siesenop U, Böhm KH.In 1993 and 1994 a highly increased occurrence of equine dermatophilosis was observed, and a study was initiated to determine phenotypic heterogeneity among 120 clinical isolates using biochemistry, antibiotic resistance profiles, membrane protein profiles and Western blotting. The biochemical examinations contained 1% equine serum in medium. Moreover, the API ZYM-test from bioMérieux was used. The biochemical reactions were suited to identify Dermatophilus congolensis but did not allow a differentiation among the various isolates. Antibiotic resistance in one or more isolates was observed ag...
Puffer BA, Watkins SC, Montelaro RC.We have identified an interaction between the equine infectious anemia virus (EIAV) late assembly domain and the cellular AP-2 clathrin-associated adapter protein complex. A YXXL motif within the EIAV Gag late assembly domain was previously characterized as a sequence critical for release of assembling virions. We now show that this YXXL sequence interacts in vitro with the AP-50 subunit of the AP-2 complex, while the functionally interchangeable late assembly domains carried by the Rous sarcoma virus p2b protein and human immunodeficiency virus type 1 p6 protein, which utilize PPPY and PTAPP ...
Clegg PD, Jones MD, Carter SD.Loss of articular cartilage, which is the most important pathological lesion occurring in osteoarthritis, has been shown to be enzymatically mediated. The matrix metalloproteinases (MMPs) are a group of enzymes which have been implicated in this degradation of articular cartilage matrix. The use of pharmacological agents to inhibit this catabolic process in the joint is a potential route for therapeutic intervention. The gelatinase MMPs, MMPs-2 and 9, were purified by affinity chromatography from equine cell cultures. The ability of phenylbutazone, flunixin, betamethasone, dexamethasone, methy...
Raidal SL, Bailey GD, Love DN.Flow cytometric techniques were developed for the evaluation of oxidative burst activity in equine peripheral blood neutrophils and lymphocytes, as well as bronchoalveolar lavage derived pulmonary alveolar macrophages and lymphocytes. The oxidation of dichlorofluorescin was measured by the increased fluorescence of cells stimulated with phorbol myristate acetate or a variety of other stimulants. Flow cytometry was a suitable method for the evaluation of the intracellular oxidation in all cell populations evaluated. Analysis was rapid and cell separation before analysis was not required. Hetero...
Tozaki T, Hirota K, Mashima S, Tomita M, Mukoyama H.A genomic clone isolated from an equine genomic library probed with an oligonucleotide (CAG)10 showed high sequence similarity to the human F18 gene and was tentatively named equine F18 gene. Because the human F18 gene is expressed in many tissues, we examined whether this equine clone was also expressed in equine tissues. The cDNA encoding equine F18 was obtained by the reverse transcriptase-polymerase chain reaction (RT-PCR) from equine thymus. The nucleotide sequence of the equine F18 cDNA (1940 bp) was determined and contained both the ATG initiation codon and a poly(A) sequence. The cDNA ...
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...
Williamson RM, Beveridge I, Gasser RB.To compare the sensitivities of three coprological techniques for the diagnosis of Anoplocephala perfoliata infection in horses and to assess the value of the methods for diagnosis of horses at risk of clinical cestodiasis. Methods: Faecal samples were collected from necropsied horses with or without A perfoliata infection and examined using one sedimentation and two different flotation methods. The coprological results were compared with worm counts performed at necropsy of the horses and the degree of mucosal damage. In addition, the efficiency of recovery of A perfoliata eggs from faeces wa...
el Hasnaoui H, el Harrak M, Tber A, Fikri A, Laghzaoui K, Bikour MH.An indirect fluorescent antibody (IFA) technique was used to screen and quantify antibodies against African horse sickness virus (AHSV) in equine sera. Results obtained with the IFA assay were compared directly with those obtained with standard complement fixation (CF) and virus neutralisation (VN) tests using horse sera from experimental studies and samples from the field. Positive fluorescent antibody titres were detected from as early as 7 days after primary vaccination and persisted for at least six months. The IFA technique offers a clear advantage over CF tests, where the antibodies are ...
Rubio C, Cubillo MA, Hooghuis H, Sanchez-Vizcaino JM, Diaz-Laviada M, Plateau E, Zientara S, Crucière C, Hamblin C.The mortality rate in susceptible populations of horses during an epizootic of African horse sickness (AHS) may be in excess of 90%. Rapid and reliable assays are therefore essential for the confirmation of clinical diagnoses and to enable control strategies to be implemented without undue delay. One of the major objectives of a recent European Union funded project was the validation of newly developed diagnostic assays which are rapid, sensitive, highly reproducible and inexpensive, for the detection of African horse sickness virus (AHSV) antigens and antibodies. The Laboratorio de Sanidad y ...
Zientara S, Sailleau C, Moulay S, Crucière C, el-Harrak M, Laegreid WW, Hamblin C.A coupled reverse transcriptase-polymerase chain reaction assay (RT-PCR) for the detection of African horse sickness virus (AHSV) dsRNA, has been developed using genome segment 7 as the target template for primers. RNA from isolates of all nine AHSV serotypes were readily detected. The potential inhibitory effects of either ethylene diamine tetra acetic acid (EDTA) or heparin on the RT-PCR were eliminated by washing blood samples before lysis of the red blood cells and storage. There was a close agreement in the sensitivity and the specificity of the RT-PCR and an indirect sandwich ELISA. Conf...
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.
Meskers SC, Dekkers HP.For mitochondrial ferricytochrome c from horse, cow and tuna and for bacterial cytochrome c-550 from Paracoccus versutus, the pressure and temperature dependence of their quenching of racemic Tb(DPA)3(3-) and Eu(DPA)3(3-) (DPA = pyridine-2,6-dicarboxylate) luminescence in aqueous solution is investigated. Of these energy transfer reactions the activation volumes (delta V#) and energies (Ea) are determined for the ranges P = 0-3 kbar and T = 15-40 degrees C. For the lambda enantiomers of Tb(DPA)3(3-) and Eu(DPA)3(3-), delta V# and Ea are almost the same for all proteins: 0.4 < or = delta V# < o...
Okumura M, Fujinaga T.To establish a sensitive and specific monoclonal antibody (MAB) against equine keratan sulfate (KS) and to develop an enzyme immunoassay for measurement of the concentration of KS in serum and synovial fluid from horses. Methods: 18 synovial fluid and 48 serum samples were obtained from clinically normal horses and horses with arthritis. Methods: BALB/c mice were immunized with chondroitinase-ABC-digested proteoglycan monomer from equine joint cartilage, and MAB were raised, using Sp2/O cells as a fusion partner. A competitive ELISA was optimized, using one of the established MAB, and KS conce...
Garbís SD, Hanley L, Kalita S.Thiazide-based diuretics are included in the list of banned drugs in the horse-racing industry. One effect of their misuse is increased urine flow, contributing to dilution of other doping agents. Their determination is essential in ensuring compliance to horse-racing regulation. This study evaluates the feasibility of using liquid chromatography/mass spectrometry (LC/MS) with electrospray and atmospheric pressure chemical ionization interfaces to analyze thiazidic diuretics in equine urine samples. Existing LC and gas chromatography/MS methods are limited in their applicability to thiazide an...
Stang BV, Koller LD.To establish baseline information on neopterin concentrations in livestock, companion and laboratory animals and identify the factors that may influence these concentrations, blood samples were taken from normal dairy cattle, horses, llamas, dogs, cats and rats of varying ages and sexes. In addition, neopterin concentrations in normal, adult equines were compared with those found in racing Thoroughbreds. There were no differences due to sex, sexual maturity, pregnancy, castration, or age. For all ages and sexes combined, mean neopterin concentrations were significantly lower in llamas (2.27+/-...
Luo Y, McNamara B, Fennell MA, Teleis DC, May L, Rudy J, Watson AO, Uboh CE, Soma LR.A rapid and sensitive method for the extraction and quantification of penicillin-G and procaine in horse urine and plasma samples has been successfully developed. The method involves the use of solid-phase extraction (SPE) for penicillin-G, liquid-liquid extraction (LLE) for procaine, and high-performance liquid chromatography (HPLC) for the quantification of penicillin-G and procaine. The new method described here has been successfully applied in the pharmacokinetic studies of procaine, penicillin-G and procaine-penicillin-G administrations in the horse.
Maury W, Oaks JL, Bradley S.Previous cell infectivity studies have demonstrated that the lentivirus equine infectious anemia virus (EIAV) infects tissue macrophages in vivo and in vitro. In addition, some strains of EIAV replicate to high titer in vitro in equine fibroblasts and fibroblast cell lines. Here we report a new cell type, macrovascular endothelial cells, that is infectible with EIAV. We tested the ability of EIAV to infect purified endothelial cells isolated from equine umbilical cords and renal arteries. Infectivity was detected by cell supernatant reverse transcriptase positivity, EIAV antigen positivity wit...
Iniguez P, Zientara S, Marault M, Machin IB, Hannant D, Cruciere C.A random hexapeptide fusion-phage library was screened to isolate phages that bind to antibodies present in horse sera positive for equine arteritis virus (EAV). Analysis of the peptide sequences displayed by isolated phages identified seven groups. 25% of the isolated phages used as antigens in an ELISA test were specifically recognised by a pool of sera which was positive for EAV in virus neutralisation test (VN). Five of these, when used as antigen in ELISA, detected greater than 50% of sera (n = 30) containing antibodies to EAV as detected by VN. When these five phages were pooled together...
Mizuguchi M, Arai M, Ke Y, Nitta K, Kuwajima K.The equilibrium unfolding and the kinetics of unfolding and refolding of equine lysozyme, a Ca2+-binding protein, were studied by means of circular dichroism spectra in the far and near-ultraviolet regions. The transition curves of the guanidine hydrochloride-induced unfolding measured at 230 nm and 292.5 nm, and for the apo and holo forms of the protein have shown that the unfolding is well represented by a three-state mechanism in which the molten globule state is populated as a stable intermediate. The molten globule state of this protein is more stable and more native-like than that of alp...
Bailey SR, Elliott J.5-hydroxytryptamine (5-HT) is a potent vasoconstrictor of equine digital arteries and veins which may play a role in the ischaemic disease, laminitis. The present investigation compared the properties of 5-HT1B/1D receptors in arteries with those in veins using isolated rings of equine digital blood vessels. The 5-HT1B/1D receptor-selective agonists, anpirtoline and sumatriptan were 17.9 and 10 times more potent and produced 4.1 and 5.6 times greater maximum contractions, respectively, in veins when compared to arteries. Other agonists tested were of equal potency and produced the same maximum...
Nerenberg C, Runkel RA, Matin SB.A simple radioimmunoassay was developed for the determination of oxfendazole in plasma. Oxfendazole N-1(3)-valerate was coupled to polylysine via a carbodiimide reaction, and antiserum was developed in rabbits after inoculation with oxfendazole--polylysine conjugate. The assay was developed so that oxfendazole could be measured directly in a 0.1-ml aliquot of diluted or undiluted plasma. With the developed procedure, 200 pg of oxfendazole/ml of plasma can be determined quantitatively. Cross-reactivity was determined for closely related compounds and metabolites. The method was used to determin...
Love LB, Choi YH, Love CC, Varner DD, Hinrichs K.Two experiments were conducted to determine the effects of storage on equine ovaries or isolated oocytes. Ovaries were collected at an abattoir and were maintained at room temperature during collection and transport (3-9h total). After arrival at the laboratory, ovaries were divided into three groups: immediate oocyte collection (control), storage at room temperature overnight (15-18 h) before oocyte collection, or storage at 4 degrees C overnight before oocyte collection. Collected oocytes were cultured in maturation medium for 24h. There was a significant increase in the proportion of oocyte...
Hoffmann SL, Seminoff K, McKemie DS, Kass PH, Knych HK.Grapiprant is a prostaglandin E2 receptor antagonist that has been found to be an effective anti-inflammatory in dogs and that is devoid of some of the adverse effects associated with traditional NSAIDs that elicit their effects through inhibition of PGE2 production. Previously published reports have described the pharmacokinetics of this drug in horses when administered at 2 mg/kg; however, pharmacodynamic effects in this species have yet to be described. The objective of the current study was to describe the pharmacokinetics and pharmacodynamics of grapiprant at a higher dose. Eight horses...
Mennella MR, Jones MR.The activity of 5'-nucleotidase (EC 1.3.5), cyclic nucleotide phosphodiesterase (EC 2.1.4.17), non-specific phosphodiesterase (EC 3.1.4.1) and ribonuclease (EC 1.7.7.16)has been investigated in the seminal plasma of whole semen and in the secretions of the seminal vesicle, prostate and epididymis of the bull, boar, ram, stallion, jackass, rabbit and man. Bull seminal plasma showed the highest activity for 5'-nucleotidase, cyclic nucleotide phosphodiesterase and ribonuclease; in contrast, stallion and jackass semen were very poor in these enzymes. Ram, rabbit and boar seminal plasma showed inte...
Waraksa E, Kowalski K, Kłodzińska E, Rola R, Ciekot J, Filipiak W, Bieńkowski T, Namieśnik J.Gamma-oryzanol (GO) has gained special attention in the equine sports industry in recent years due to its touted properties, including the fact that it may cause anabolic effects on muscle growth and reduce fatigue. Many manufactures offer supplements containing GO as a naturally occurring anabolic substance; however, some producers do not declare its presence in product compositions. Taking into consideration the touted properties of GO, its ambiguous effectiveness and the open character of the Prohibited Substances List established by the Fédération Equestre Internationale, there is an urg...
Rivero JL, Serrano AL, Henckel P.Biopsies of the gluteus medius muscle were taken at three different depths from 36 endurance horses aged 8.42 +/- 2.85 years and of both sexes. Twenty of the horses were considered to be excellent performers on the basis of the mean speed of their three fastest records in endurance events over the previous two or three years, whereas 16 were moderate performers. The biopsy samples were analysed for the activities of the enzymes citrate synthase (an indicator of citric acid cycle activity), 3-OH-acyl-CoA-dehydrogenase (an indicator of lipid oxidation) and lactate dehydrogenase (an indicator of ...
Hifumi T, Akioka K, Tanaka T, Miyoshi N.Alveolar echinococcosis, which is caused by a larval-stage infection of Echinococcus multilocularis, is a zoonosis with public health importance. Recently, alveolar echinococcosis in slaughtered horses has been reported in Japan and Poland. In terms of public health, a highly sensitive and specific diagnostic method is essential for early detection during meat inspection. In this study, the loop-mediated isothermal amplification (LAMP) assay was developed and validated to target the mitochondrial cytochrome b (cob) gene of E. multilocularis. Forty-one hepatic solid nodules obtained from each h...
Chang CK, Ward B, Ebina S.Carbon monoxide- and oxygen-binding rates and affinities were measured for horse heart myoglobins reconstituted with synthetic hemes lacking peripheral methyl and vinyl groups. There is an apparent correlation between heme size and ligand specificity, i.e. larger m values (ratios of CO vs O2 association rates, l'/k') with smaller hemes. However, this correlation broke down with the most dealkylated heme. This is interpreted as resulting from protein conformational changes altering the steric crowdedness at the O2-binding site. Spectral properties and autoxidation rates also corroborate this vi...
Cozzi B.A combined ultrastructural and immunocytochemical study was performed on the pineal gland of the horse in order to identify the cell types present and describe their characteristics. Comparisons have been made with other mammals. Two main cell types are present: pinealocytes and glial cells. Pinealocytes display different degrees of electron density in the nucleus and the cytoplasm, yet no ultrastructural feature supports the idea of separate populations. Putative secretory materials are stored in vesicles related to the Golgi apparatus. A variety of electron-dense bodies are present in the cy...
Ramos MI, Hermosura ME, Nakabayashi T.Horse, calf and bovine serum were successfully used as human serum substitutes in the in vitro cultivation of Plasmodium falciparum. Positive results were obtained only after gradually adapting the parasites to the substitute serum. Adapted lines were established within 4-5 weeks. 10% horse serum was observed to be the best substitute with growth rates comparable or even surprising that obtained in human serum. Pure calf or bovine serum supported stable growths of 20-30% less which was enhanced to comparable levels after addition of 1% glucose-peptone to the medium. Direct transfers of adapted...
Janda J, Lehmann M, Luttmann W, Marti E.Interleukin-3 is a growth and differentiation factor for various hematopoietic cells. IL-3 also enhances stimulus-dependent release of mediators and cytokine production by mature basophils. Function of IL-3 has not been studied in horses because of lack of horse-specific reagents. Our aim was to produce recombinant equine IL-3 and test its effect on sulfidoleukotriene and cytokine production by equine peripheral blood leukocytes (PBL). Equine IL-3 was cloned, expressed in E. coli and purified. PBL of 19 healthy and 20 insect bite hypersensitivity (IBH)-affected horses were stimulated with Culi...
Brown SA, Dusza K, Boehmer J.A relation exists between colloid osmotic pressure (pi) and serum total protein concentration; equations describing this relation have been used to determine a calculated value for pi. However, the relation between total protein concentration and pi is altered by the method used to measure protein and by changes in the ratio of concentrations of albumin (A) to globulin (G). We developed nomograms for estimating pi from A and G concentrations, using samples obtained from clinically normal animals and compared the accuracy of these nomograms with that of previously described equations relating p...
Khoshtariya DE, Dolidze TD, Shushanyan M, van Eldik R.Horse muscle myoglobin (Mb) was tightly immobilized at Au-deposited ~15-Å-thick mixed-type (1:1) alkanethiol SAMs, HS-(CH₂)₁₁-COOH/HS-(CH₂)₁₁-OH, and placed in contact with buffered H₂O or D₂O solutions. Fast-scan cyclic voltammetry (CV) and a Marcus-equation-based analysis were applied to determine unimolecular standard rate constants and reorganization free energies for electron transfer (ET), under variable-temperature (15-55 °C) and -pressure (0.01-150 MPa) conditions. The CV signal was surprisingly stable and reproducible even after multiple temperature and pressure cycl...
Viljanto M, Kaabia Z, Taylor P, Muir T, Habershon-Butcher J, Bailly-Chouriberry L, Scarth J.Boldenone is an anabolic-androgenic steroid that is prohibited in equine sports. However, in certain situations, it is endogenous or is believed to be formed by microbes in urine, and therefore, an approach for the differentiation is required. Following the identification of Δ1-progesterone and 20(S)-hydroxy-Δ1-progesterone as potential biomarkers of microbial activity, the presence of six steroids was investigated in the postrace urine of castrated male horses (geldings, n = 158). In line with endogenous findings from several other species when ultrasensitive methods are employed, bolde...
Taddei N, Buck M, Broadhurst RW, Stefani M, Ramponi G, Dobson CM.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...
Madry MM, Spycher BS, Kupper J, Fuerst A, Baumgartner MR, Kraemer T, Naegeli H.Compared to blood or urine, drugs can be detected for much longer periods in the long hair of horses. The aim of this study was to establish and validate a highly sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the detection and quantification of frequently prescribed opioids, sedatives and non-steroidal anti-inflammatory agents in the mane and tail hair of horses. Based on an average growth rate of about 2 cm per month, times of administration reported by horse owners or veterinary physicians were related to drug localizations in hair. Hair samples were collecte...
Gao X, Jiang CG, Han XE, Zhao LP, Shen RX, Xiang WH, Zhou JH.To elucidate the function of the S2 gene in equine infectious anemia virus (EIAV) and its role in the attenuation of the Chinese attenuated EIAV vaccine strains, the S2 in the EIAV vaccine strain EIAV (FDDV) was reverse-mutated and the in vitro replication character of the resultant virus was evaluated. Based on the sequence variation of the S2 gene between the EIAV virulent strains and vaccine strains, all the four vaccine-specific sites in the S2 of an EIAV(FDDV) infectious clone, pFDDV3-8, were reverse-mutated to the sequences of the virulent strain EIAV(DV115). The reverse-mutated molecula...
Donné-Op den Kelder GM, de Haas GH, Egmond MR.At alkaline pH porcine pancreatic phospholipase A2 is known to bind two Ca2+ ions per protein molecule. One Ca2+ ion is strongly bound to the active site and is essential for enzyme activity. A second Ca2+ ion binds more weakly to the protein and improves the affinity of the enzyme for lipid-water interfaces severalfold at high pH values. A group having a pK around 6 controls enzyme binding to lipid-water interfaces in the absence of Ca2+. By use of proton titration techniques this group is now identified to be a carboxylate having an abnormally high pK. Its pK shifts to a value around 4.5 in ...
Heiskanen ML, Pirhonen A, Koskinen E, Mäenpää PH.The role of various environmental conditions on sperm motility and ATP content was investigated by incubating raw and washed spermatozoa collected with an open-ended artificial vagina from 10 stallions in various biological and artificial media under different atmospheric conditions. Spermatozoa did not survive for more than 12 h when kept unextended in the original seminal fluid in any circumstances. The most favourable media tested for long-term sperm survival were Kenney's medium or Kenney's medium supplemented with 10 mM-theophylline and 10 mM-Hepes, pH 7.2. Centrifugation and slow cooling...
Southwood LL, Gassert T, Lindborg S.It is the impression of some surgeons that geriatric horses have a lower survival rate compared to mature nongeriatric horses following colic surgery. One possible reason for this is that geriatric horses may be more critically ill at admission and have more severe disease than mature nongeriatric horses. Objective: To compare admission historical, physical examination and laboratory data for geriatric and mature nongeriatric horses referred for signs of colic. Methods: Medical records of horses admitted with a presenting complaint of colic between 2000 and 2006 were reviewed. Geriatric horses...
McDonald TL, Burger D.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...
Salami F, Ghodrati M, Parham A, Mehrzad J.Bone marrow mesenchymal stem cells (BM-MSCs), as multipotent cells with self-renewal and plastic-adherent properties, have immunomodulatory effects on immune cells, including neutrophils. These cells are in close proximity in bone marrow (BM) sinusoids with non-multiplicative immature neutrophils. BM-MSCs exert their immunomodulatory effects on adjacent cells both directly (cell-to-cell contact) and indirectly (secretion of soluble factors). The aim of this study was to evaluate the effect of equine bone marrow mesenchymal stem cells (BM-MSCs) on the expression of some pro- and anti-apoptotic ...
Johnston JK, Morris DD.Sixty-eight horses with colic caused by small intestinal disease were allotted into 2 groups of 34 on the basis of recorded findings during exploratory celiotomy, necropsy, or response to medical treatment alone. Signalment, history, physical examination findings, and laboratory findings were compared between the group of horses with small intestinal obstruction and the group with duodenitis/proximal jejunitis. A significantly greater proportion of horses with duodenitis/proximal jejunitis were older than 2 years old (P less than 0.05). Differences in sex or breed distribution, or in seasonali...
Karakka Kal AK, Nalakath J, Kunhamu Karatt T, Perwad Z, Mathew B, Subhahar M.The detection and separation of medetomidine enantiomers from the complex biological matrices poses a great analytical challenge, especially in the field of forensic toxicology and pharmacology. Couple of researchers reported resolution of medetomidine using protein-based chiral columns, but the reported method is quiet challenging and tedious to be employed for routine analysis. This research paper reported a method that enables the enantio-separation of medetomidine by using polysaccharide cellulose chiral column. The use of chiralcel OJ-3R column was found to have the highest potential for ...
Deng L, Li Z, Tang C, Han Y, Zhang L, Liao Q.Equine pregnancy is currently diagnosed by rectal palpation, ultrasonographic examination, or by measuring changes in hormones in the blood. In the present study, we identified proteins that are differentially expressed in the sera of early pregnant and non-pregnant mares in order to develop a novel method for diagnosing equine pregnancy. Serum samples were obtained from 18 adult mares, pregnancy at day 32 after ovulation (n = 9) and in diestrus (n = 9). Proteomic analysis of the samples was conducted using liquid chromatography-electrospray ionization-tandem mass spectrometry. We iden...
Schnabel CL, Babasyan S, Freer H, Wagner B.Only few quantitative reports exist about the concentrations and induction of immunoglobulin A (IgA) in mucosal secretions of horses. Despite this, it is widely assumed that IgA is the predominant immunoglobulin on mucosal surfaces in the horse. Here, two new monoclonal antibodies (mAbs) against equine IgA, clones 84-1 and 161-1, were developed and characterized in detail. Both IgA mAbs specifically bound monomeric and dimeric equine IgA in different applications, such as Western blots and fluorescent bead-based assays. Cross-reactivity with other equine immunoglobulin isotypes was not observe...
Todhunter RJ, Wootton JA, Altman N, Lust G, Minor RR.Collagen type I and type II were purified from equine flexor tendon and articular cartilage, respectively. Equal amounts of these collagens were cleaved with cyanogen bromide, and 11 mixtures containing increasing proportions of type II collagen were separated in seven identical sodium dodecyl sulfate-polyacrylamide gels. The density of bands was measured in wet gels and the peak areas were used to form six ratios of peptide bands that had polynomial relationships with the known proportions of type I and type II collagen in the mixtures. Calibration curves for determining the proportion of typ...
Murray JA, Longland AC, Moore-Colyer MJ, Dunnett C.A series of experiments was conducted to determine the effects of a fibrolytic enzyme preparation (enzyme 1; E1) on the in vitro fermentation of lucerne incubated with equine faecal inocula. In experiment 1, high-temperature-dried (HT) lucerne was treated with five levels of E1 (0 to 2.4 ml/g DM) and incubated at 50 degrees C for 20 h. Samples then received a simulated foregut digestion (SFD) treatment before DM and NSP analysis. In experiment 2, HT lucerne was treated with the same enzyme levels used in experiment 1. Samples were then split into two groups; plus or minus an SFD treatment befo...
Dai X, Hilsen RE, Hu WG, Fulton RE.An electrochemiluminescence (ECL) immunoassay, incorporating chemically biotinylated and ruthenylated antibodies down-selected from a panel of monoclonal and polyclonal reagents, was developed to detect and identify Venezuelan equine encephalitis virus (VEEV). The limit of detection (LOD) of the optimized ECL assay was 10(3)pfu/ml VEEV TC-83 virus and 1 ng/ml recombinant (r) VEEV E2 protein. The LOD of the ECL assay was approximately one log unit lower than that of a sandwich enzyme-linked immunosorbent assay (ELISA) incorporating the same immunoreagents. Repetition of ECL assays over time and...
Crews HM, Dean JR, Ebdon L, Massey RC.The speciation of cadmium in retail pig kidney has been examined by size-exclusion chromatography (SEC) coupled directly to inductively coupled plasma mass spectrometry (ICP-MS). Approximately 35% of the cadmium from uncooked kidney was soluble after aqueous extraction at pH 8 and SEC - ICP-MS revealed three discrete peaks whose retention times corresponded to estimated relative molecular masses of 1.2 x 10(6), 7.0 x 10(4) and 6 x 10(3)-9 x 10(3). In the cooked kidney, 35% of the Cd was soluble and was all associated with a peak of a relative molecular mass (Mr) of 6 x 10(3)-9 x 10(3). After s...
