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.
Sanada Y, Noda H, Nagahata H.A fluorometric assay was applied to evaluate blastogenesis of equine lymphocytes. Optimal culture conditions were as follows; concentrations of phytohaemagglutinin-P (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) were 1 microgram/ml, 40 micrograms/ml and 10 micrograms/ml, respectively, when 5 X 10(5) lymphocytes were incubated with culture medium containing 20% pooled horse serum (PHS) for 120 hours. The relative mean stimulation index of healthy non-pregnant mares were 5.107 +/- 0.323 (M +/- SE) with PHA, 4.019 +/- 0.183 with Con A and 3.610 +/- 0.131 with PWM. Sequentially the blas...
Truax RE, Powell MD, Montelaro RC, Issel CJ, Newman MJ.A rapid and simple technique for the cryopreservation and recovery of equine mononuclear cells was developed. Buffy-coat leukocytes were frozen in autologous plasma containing 10% DMSO and mononuclear cells were recovered by gradient sedimentation using a standard Ficoll-Hypaque purification procedure. The total numbers of mononuclear cells recovered from cryopreserved samples were 94%-82% of those recovered from fresh blood samples. The functional capabilities of the mononuclear cells from cryopreserved buffy coat preparations were compared with those of mononuclear cells from fresh samples b...
Adolf GR, Traxler E, Maurer-Fogy I.Equine interferon-beta 1 (EqIFN-beta 1) was purified from extracts of recombinant Escherichia coli by sequential chromatography on hydroxylapatite, anion-, and cation-exchangers. The resulting protein was greater than 98% pure as determined by sodium dodecylsulfate gel electrophoresis, gel permeation HPLC, and reverse-phase HPLC. Amino-terminal amino acid sequencing revealed that essentially all molecules contained an additional amino-terminal methionine. The specific antiviral activity of EqIFN-beta 1 determined on equine dermal fibroblasts challenged with vesicular stomatitis virus (VSV) was...
Rej R, Rudofsky U, Magro A, Prendergast J.Aminotransferase activities were measured in the serum of two- to three-year-old Thoroughbred fillies and colts during a four week period of peak training for flat racing. Aspartate aminotransferase (AspAT, EC 2.6.1.1), mitochondrial aspartate aminotransferase (m-AspAT) and alanine aminotransferase (AlaAT, EC 2.6.1.2) activities in serum were measured and the relative proportions of apoenzyme and holoenzyme were determined. The aminotransferase activities were increased only slightly immediately following exercise. This small and immediate post exercise increase in activity did not vary greatl...
McDowell KJ, Sharp DC, Fazleabas AT, Roberts RM.Conceptuses were obtained from pony mares on each day of pregnancy between Days 12 and 28, and on Days 39, 45, 65 and 100. Endometrium was obtained from mares at Days 12, 14, 16, 18, 39, 45, 65 and 100 of pregnancy, and from non-pregnant mares during anoestrus, during transition into the breeding season, at oestrus, or during dioestrus. Tissues were incubated in vitro for 24 h with L-[3H]leucine. Proteins synthesized and released into the culture medium were analysed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and fluorography. Conceptuses obtained before Day 14 after ovul...
Araújo AC, Salvadori MC, Velletri ME, Camargo MM.The possibility of false negative results from TLC when a diuretic is administered concomitantly with flunixin was studied. Samples were subjected to solvent extraction from acidic aqueous solutions; duplicate samples were also subjected to alkaline hydrolysis at pH 12.5. The internal standard was flufenamic acid. The quantification of flunixin was performed by HPLC and the results confirmed by GC/MS. The data show that furosemide influences the urinary concentration of flunixin.
Tulamo RM, Saari H, Konttinen YT.Concentration of hyaluronate (HA) in equine serum was determined by a recently developed specific radioassay. The mean +/- SD HA concentration in equine serum was 288 +/- 145 micrograms/L, was age dependent, and varied widely between horses (range, 190 to 760 micrograms/L). Light or moderate exercise increased serum HA concentration from baseline values by 1.5- to 3-fold. In all horses, serum HA concentration returned to or below the original resting values 1 and 2 hours after exercise.
Ballagi-Pordány A, Klingeborn B, Flensburg J, Belák S.Primers and probes were selected from the gene encoding glycoprotein 13 (gp 13) of equine herpesvirus 1 (EHV-1). The polymerase chain reaction (PCR) was run on infected and noninfected cultured cells and on 63 specimens from 29 aborted equine fetuses. The results were evaluated by electrophoresis and dot-blot hybridization using an oligonucleotide probe labeled with biotin. In the infected samples electrophoresis showed a PCR product of about 280 base pairs. The dot-blot hybridization confirmed that this product contained EHV-1 DNA sequences. PCR took 4 h and hybridization another 14 h; the re...
Bell CW, Boyle DB, Whalley JM.Transcript mapping of the equine herpesvirus 1 (EHV-1) glycoprotein B (gB) gene homologue by Northern blot, S1 nuclease and primer extension analyses indicated that two overlapping transcripts of 3.4 and 4.6 kb originated from the same strand and were transcribed from left to right between coordinates 0.40 and 0.43 of the EHV-1 genome. The 3.4 kb transcript encoded EHV-1 gB and the 5' RNA terminus was located approximately 30 bases downstream from a probable TATA element. The coding region of the gB gene homologue was reconstructed from two subclones using oligonucleotide mutagenesis and inser...
Monzón CM, Mancebo OA, Roux JP.In a total of 165 blood samples from horses in the Province of Formosa (Argentina), the diagnosis for equine trypanosomiasis (T. evansi) was made using Giemsa-stained smears (GSS), wet blood films (WBF), Strout's concentration method (SCM), haematocrit centrifuge technique (HCT), buffy coat method (BCM) and mouse inoculation of blood (MBI). Trypanosoma evansi was demonstrated in 52 samples. Mouse inoculation gave a sensitivity of 88.2%; HCT 71.1%; BCM 63.4%; WBF 53.8%; SCM 46.1% and GSS 45.6%. No single method alone was totally effective. The haematocrit centrifuge technique, mouse inoculation...
Ruiz Silva BE, Burtnick LD.Gelsolin can be purified from horse blood plasma by treating the plasma sequentially with an anion-exchange medium in the presence and then the absence of free Ca2+. The purified gelsolin migrates as a 90-kilodalton protein on electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate. It has an absorption coefficient of 1.4 mL/(mg.cm) and is similar in amino acid composition to other plasma gelsolins. Horse plasma gelsolin has an intrinsic sedimentation coefficient of 4.8S and a Stokes' radius of 3.8 nm. Hydrodynamic calculations suggest it to be a rather globular protei...
Engen RL, Clark CL.High-performance liquid chromatography (HPLC) was used to determine the phospholipid (PL) composition of ovine, equine, bovine, porcine, and canine RBC membranes. Procedural modifications of established techniques provided for separation of 7 PL within a 15- to 20-minute sample run. Significant (P less than 0.05) differences were detected in RBC membrane PL composition among the various species. The concern for physiologic properties associated with hemolysis and/or sedimentation rate must include evaluation of differences in the PL bilayer structure.
Kline KH, Bechtel PJ.The purpose of this study was to investigate metabolic changes in equine muscle from birth to 1 yr of age. Duplicate biopsies from the middle portion of the gluteus medius were obtained from a depth of 2 cm beneath the superficial fascia at 1 day, 7 days, 1 mo, 3 mo, 6 mo, and 1 yr of age in 11 quarter horses and at 1 day, 3 mo, 6 mo, and 1 yr of age in 5 Standardbreds. Muscle enzyme activities determined were citrate synthase, 3-hydroxyacyl-CoA dehydrogenase, phosphorylase, and lactate dehydrogenase. Percent fast-twitch, fast-twitch high oxidative, and slow-twitch oxidative fiber types were d...
Noiman S, Yaniv A, Sherman L, Tronick SR, Gazit A.The pattern of expression of the equine infectious anemia virus (EIAV) genome in a persistently infected canine cell line was determined. Five EIAV-specific transcripts (8.2, 5.0, 4.0, 2, and 1.8 kilobases [kb]) were detected by using subgenomic restriction enzyme fragments of EIAV DNA and EIAV-specific oligonucleotides as probes. The 8.2-kb mRNA could be shown to represent viral genomic RNA, whereas the smaller transcripts were generated by splicing events. Evidence was obtained that indicated that each subgenomic RNA species shared a common 5'-splice donor. The 5.0-kb mRNA was found to be ex...
Kashi Y, Tikochinsky Y, Genislav E, Iraqi F, Nave A, Beckmann JS, Gruenbaum Y, Soller M.Southern blots of genomic DNA from a variety of species digested by restriction endonucleases having a four-bp specificity, were probed with a bovine genomic clone consisting of seven tandem poly-TG stretches separated by a 29bp linker sequence. Highly variable DNA 'fingerprint' patterns were obtained in chicken, sheep, and horse, moderately variable DNA 'fingerprints' in mouse and man, and a monomorphic pattern in Drosophila. In chicken, horse and man a (TG)10 synthetic oligonucleotide probe gave results identical to those given by the bovine probe. Furthermore, in chicken the DNA fingerprint...
Kotze SH, Van Aswegen G.The ileocaecal junctions of 5 horses and 2 donkeys were examined by using antisera to the following peptides: somatostatin, glucagon, gastrin, neurotensin, vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), calcitonin gene-related peptide (CGRP), substance P (SP) and neuropeptide Y (NPY). Antisera to somatostatin, neurotensin and NPY demonstrated endocrine cells in the ileal- and caecal parts of the ileocaecal junction, while immunoreactivity for glucagon was demonstrated in endocrine cells of the ileal part only. Nerve cell bodies showing immunoreactivity to SP, VIP, CGR...
Morris DD, Moore JN, Fischer K, Tarleton RL.A study was performed to determine whether equine macrophages produce tumor necrosis factor (TNF) activity in vitro in response to endotoxin and to study the effects of endotoxin concentration and incubation time on the amount of TNF produced. Equine peritoneal macrophages were isolated and cultured in vitro for 2, 6, 12, or 24 hr in tissue culture media containing 1) no additive (nonstimulated control), 2) endotoxin (0.5 ng/ml, 5 ng/ml, or 5 micrograms/ml), or 3) the calcium ionophore A23187 (0.95 microM). The supernatant media concentrations of TNF activity were determined by an in vitro cyt...
Egberink HF, Ederveen J, Montelaro RC, Pedersen NC, Horzinek MC, Koolen MJ.Feline immunodeficiency virus (FIV) grown in cat lymphocyte and thymocyte cultures was labelled with L-[35S]methionine or [3H]glucosamine and virus-coded proteins were identified using immunoprecipitation. Polypeptides with apparent Mr values of 15K, 24K, 43K, 50K, 120K and 160K were detected. An additional polypeptide of 10K was detected by Western blot analysis. The two highest Mr species sometimes appeared as one band, of which only the 120K polypeptide was glycosylated. In the presence of tunicamycin gp120 was no longer detectable and a non-glycosylated precursor of 75K was found instead. ...
Auer DE, Ng JC, Seawright AA.Polymorphonuclear leukocytes (PMNLs) were isolated from an inflammatory exudate induced in the intercarpal joints of horses by an administration of carrageenin. Their superoxide production at rest and following stimulation with either serum-treated zymosan (STZ) or phorbol myristate acetate (PMA) was measured by cytochrome-c reduction. Stimulation of the cells increased the cytochrome-c reduction 10-15 times that of resting cells. The maxima were 20 nmol of reduced cytochrome-c per 10(6) cells per ml at 120 min (STZ) and 35 nmol of reduced cytochrome-c per 10(6) cells per ml at 60 min (PMA). T...
May SA, Hooke RE, Lees P.Equine interleukin-1 has been produced from peripheral blood monocytes by stimulation with E. coli lipopolysaccharide. Sephacryl S200 gel filtration revealed a molecular weight of 17-18 kD. Chromatofocusing of the 17-18 kD peak identified four active fractions. Two major peaks were detected at pH 6.7 and pH 7, with smaller peaks at pH 6.3 and pH 5.9. The pI 7 molecule is probably the equine form of IL-1 beta.
Mollerach-Gobbi B, Retegui LA, Peña C.The immunochemical behavior of several fragments of equine growth hormone (eGH) was examined using competitive binding assays with antibodies (Abs) to eGH obtained from different sources. Antigenicity was detected within the sequences 5-72 and 73-123 by rabbit Abs to eGH and by three mouse monoclonal antibodies (MAbs) produced by using bovine growth hormone as immunogen, but showing heteroclitic properties towards eGH. The polyclonal Abs to eGH also recognized as immunoreactive two smaller peptides corresponding to the amino acid residues 52-72 and 110-123. By contrast, the heteroclitic Abs to...
Ekfalck A.A method was developed for separating different layers of the matrix of the equine hoof wall by dissection, and the layers were then analyzed with respect to their amino acid composition. The results were used to compare the biochemistry of hard keratinization (e.g., in the hoof wall matrix) and soft keratinization (e.g., in the epidermis of the skin). Hard keratinization differed from soft keratinization not only by its previously well known high incorporation of cystine, but also by considerable incorporation of tyrosine and threonine into the outer layers of the keratogenous zone and by the...
Baker TS, Newcomb WW, Booy FP, Brown JC, Steven AC.Cryoelectron microscopy and three-dimensional computer reconstruction techniques have been used to compare the structures of two types of DNA-free capsids of equine herpesvirus 1 at a resolution of 4.5 nm. "Light" capsids are abortive, whereas "intermediate" capsids are related to maturable intracellular precursors. Their T = 16 icosahedral outer shells, approximately 125 nm in diameter, are indistinguishable and may be described in terms of three layers of density, totalling 15 nm in thickness. The outermost layer consists of protruding portions of both the hexon and the penton capsomers, ris...
Shams el Din HE, el Nasri M.Seventeen isolates (4.27%) were recovered from 398 samples. Twelve isolates (4%) were obtained from 300 donkey nasal swabs, three (4.3%) and two (6.89%) isolates were recovered from 69 horse nasal swabs and 29 mare uterine washings, respectively. Nine isolates were lost during storage at -20 degrees C and the remaining eight were identified as mycoplasmas and their biological, biochemical and serological reactions were investigated. The isolates could be divided into two groups on the basis of glucose fermentation and arginine hydrolysis. The first group neither fermented glucose nor hydrolyse...
Adam A, Ong H, Sondag D, Rapaille A, Marleau S, Bellemare M, Raymond P, Giroux D, Loo JK, Beaulieu N.A monoclonal antibody was synthesized in mouse against the O-(3-carboxypropionyl) derivative of albuterol linked to bovine serum albumin. Isotyping of this material revealed the IgG1 class characterized by an affinity constant of 1.03 nM-1 and a density of sites of 0.55 nM. This antibody was found specific as its cross-reactivity to structurally related molecules was less than 1% except for clenbuterol (75%). A radioimmunoassay was set up with culture supernatant (final dilution 1/1000) and [3H] albuterol. The calibration curve was characterized by a maximum binding of 28%, an ED50 of 1.15 pmo...
Schmid DO, Ek N, Braend M.A silent allele in the transferrin locus (Tf) was observed in a Thoroughbred mare and in five of her offspring from three different matings. Evidence for the silent allele was obtained by quantitative immunodiffusion studies.
Bowling AT, Dileanis S.The C3 polymorphism of equine serum or plasma revealed by agarose gel electrophoresis can be diagnosed with protein stain following acid protein fixation. In addition to the three alleles previously described (C31, C32, C33), a fourth allele (C34) was found. Population data for 25 domestic breeds and Equus przewalskii are presented.
George JW, O'Neill SL.Most hand-held medical refractometers have internal scales that limit protein measurement to results >/=2.5 g/dL. Tables for conversion of refraction (r) to protein concentration for values as low as 0.1 g/dL were published in the 1960s, but their accuracy for use on body fluids has not been established. The purpose of this study was to assess the reliability of body cavity fluid protein determination by refractometry. We compared the protein concentration of 25 body cavity fluids as determined by 2 Goldberg type hand-held refractometers with results obtained by the biuret method. Published...
Hirano F, Imamura S, Sasaki Y, Takikawa N, Sawata A, Yamamoto A, Uchiyama M, Shimazaki Y, Kojima A, Nagai H.To establish the first National Veterinary Assay Laboratory (NVAL) equine tetanus antitoxin reference standard for veterinary use, we manufactured vials of a candidate antitoxin. These were quality tested for moisture content, vacuum, colour, clarity, and the presence of foreign objects. Ultimately, 115 quality-controlled vials were prepared. To estimate the antitoxin potency of the candidate standard, three different laboratories conducted parallel line assays alongside the existing antitoxin standard. These potency estimates ranged from 38 to 42 IU. This activity was maintained for two years...
Sepulveda MF, Greenaway EC, Avella M, Goode NT, Cunningham FM.To determine if protein kinase C (PKC) regulates equine eosinophil function. Methods: Blood eosinophils were obtained from healthy ponies. Methods: IL-5- and histamine-induced adherence to serum-coated plastic was measured as the eosinophil peroxidase content of adherent cells and serum treated zymosan (STZ)-and IL-5-induced superoxide production by the reduction of cytochrome C. Eosinophil PKC activity was quantitated as the rate of transfer of (32)P from ATP to substrate. The effects of Ro31-8220 (isotype non-selective PKC inhibitor), Go6976 (conventional PKC inhibitor), and rottlerin (PKCde...
Bridges CG, Ledger N, Edington N.Ponies, without evidence of previous exposure to Equine herpes virus-1 (EHV-1), were experimentally infected with EHV-1 subtype 2 and investigated for lymphocyte transformation to virus-infected cell polypeptides, as shown by separation with gel electrophoresis. Animals made significant responses to Western blot fractions that corresponded to molecular weights of approximately 30,000, 40,000-45,000, 60,000-65,000, 80,000-95,000 and 100,000-140,000 MW. These molecular weight ranges correlated with the positions of major EHV-1 subtype 2 glycoproteins that were found at migration distances approx...
Hamza E, Cosandey J, Gerber V, Koch C, Unger L.MicroRNAs (miRNAs) have been proposed as biomarkers for equine sarcoid (ES) disease. In this study, the suitability of three whole blood miRNAs to diagnose ES and to predict and monitor the outcome of therapy was explored. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), expression levels of eca-miR-127, eca-miR-379, and eca-miR-432 in whole blood of ES-affected equids before and at least one year after therapy were compared to those of unaffected control equids. Associations of age, sex, species, diagnosis, and therapy outcome with miRNA expression levels were exa...
Ousey JC, Delclaux M, Rossdale PD.Three strip test kits which measure calcium and/or magnesium in mares' mammary secretions were compared and assessed for their accuracy in:- 1) measuring these electrolytes compared to standard laboratory measurements, and 2) predicting the time of parturition. Daily samples of mammary secretions were obtained pre-partum from 35 Thoroughbred mares and allocated a score. Measurement of mammary secretion calcium and/or magnesium by the strip tests and laboratory methods correlated significantly (P less than 0.001). The results obtained for prediction of parturition were analysed by two methods. ...
Visser S, Jenness R, Mullin RJ.Three groups of casein components were isolated from horse milk. Group I is almost insoluble at acid and neutral pH, and is rather heterogeneous on alkaline gels with or without sodium dodecyl sulphate. Group II shows strong similarity to beta-casein from other species, as concluded from its amino acid composition and its N- and C-terminal sequences. This group consists of five electrophoretically distinguishable forms, all containing ester phosphate groups but no carbohydrate. Group III is composed of C-terminal fragments of the beta-like (group II) fraction and probably arises from the actio...
Wong KS, Cheung HW, Choi TLS, Kwok WH, Curl P, Mechie SC, Prabhu A, Wan TSM, Ho ENM.Recent advances in label-free quantitative proteomics may support its application in identifying and monitoring biomarkers for the purpose of doping control in equine sports. In this study, we developed a workflow of label-free quantitative proteomics to propose plasma protein biomarkers in horses after administration with krypton (Kr), a potential erythropoiesis-stimulating agent. Plasma proteomes were profiled by using nanoliquid chromatography-high-resolution mass spectrometry. An in-house mass spectral library consisting of 1121 proteins was compiled using samples collected from geldings (...
Fu ZF, Denby L, Lien DH, Robinson AJ.An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies against equine herpesvirus type 2 (EHV-2) in equine sera. The optimal conditions of antigen concentration, and serum and conjugate dilutions were established by chequerboard titrations. When the standard ELISA test was used for titration of test sera, it was found to give titres approximately 1500 times higher than those obtained in the virus neutralization (VN) test, and a correlation coefficient of 0.815 was obtained between these two tests on 42 equine sera. All the positive serum samples by ...
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...
Gray BP, Teale P, Pearce CM.Analysis of equine plasma samples to detect the abuse of anabolic steroids can be complicated when the parent steroid is endogenous to the animal. Anabolic steroids are usually administered intramuscularly as synthetic esters and therefore detection of the exogenous esters provides unequivocal proof of illegal administration. An ultra high performance liquid chromatography tandem mass spectrometric (UPLC-MSMS) method for the analysis of esters of testosterone (propionate, phenylpropionate, isocaproate, and decanoate) and boldenone (undecylenate) in equine plasma has been developed. Esters were...
Durham AE.Thyrotropin releasing hormone (TRH) stimulation testing is often used to support a diagnosis of pituitary pars intermedia dysfunction (PPID) in horses although it is unclear whether or not repeat TRH stimulation testing post-treatment is a valid means of assessing response to medical therapy. Laboratory submissions from 64 suspected equine PPID cases were examined including the initial pre-treatment TRH stimulation test and a follow up test within 100 days of starting medical therapy with pergolide. In a subset of cases, further follow-up tests were examined beyond 100 days of starting treatme...
Ledeck J, Dufour P, Evrard É, Le Goff C, Peeters S, Brutinel F, Egyptien S, Deleuze S, Cavalier É, Ponthier J.This study describes 17-β-estradiol (E2), estrone (E1) and estrone-sulfate (E1S) concentrations between 4 and 11 months in healthy equine pregnancies of two different breeds using Liquid Chromatography coupled to Mass-Spectrometry (LC-MS). In 2 stud-farms including 15 Spanish PureBred (SPB) and 11 Showjumping (SJ) types mares, combined thickness of the uterus and the placenta (CTUP) was measured and blood was sampled monthly between 4 and 11 months of gestation. Concentrations of E2, E1 and E1S were assayed with LC-MS in mares with normal CTUP. Effects of breed, day of pregnancy and mare's pa...
Zhang XY, Zhu FX, Robinson NE.We examined the effect of activation of beta 2-adrenoceptor (AR) by isoproterenol (ISO) on acetylcholine (ACh) release evoked by electrical field stimulation (EFS: 20 V, 0.5 Hz, 0.5 msec) from cholinergic nerves in five regions of equine airways. We also tested if the effect of ISO was dependent on epithelium or prostanoids by examining the effect of ISO on ACh release in the presence and absence of epithelium or cyclooxygenase inhibition. Trachealis strips or bronchial rings were preincubated for 60 min with 10(-7) M atropine, 10(-6) M neostigmine, and 10(-5) M guanethidine. The ACh amount wa...
Dumasia MC, Houghton E, Jackiw M.After homogenization of testicular tissue from stallions aged 1, 2 and 5 years, the unconjugated and conjugated steroids were isolated by a combined solvent-solid extraction procedure. The conjugates were further separated into glucuronides and sulphates by chromatography using Sephadex LH-20. After enzyme hydrolysis and solvolysis of the respective conjugate classes, the three extracts, unconjugated steroids, aglycones and solvolysed sulphates, were purified by chromatography using Kieselgel 60H columns. Five fractions were resolved from each extract; an aliquot of each fraction was derivatiz...
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...
Akköse M, Karabulut E, İnal Ş, Dik GÇ, Özbeyaz C, Kaya U, Çam M, Topal B.The aim of this study was to evaluate changes in the serum immunoglobulin G (IgG) and serum total protein (STP) concentrations and serum Brix percentages of neonatal Arabian foals during first 3 weeks of life. Blood samples were collected from 12 apparently healthy foals by jugular venipuncture at birth and at 12-hours, 24-hours, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 and 21 days of age. Serum IgG and STP concentrations and Brix percentages were measured by the radial immunodiffusion assay, and digital STP and Brix refractometers, respectively. Based on the serum IgG concentrations measured at 24 h, ...
Bijault C, Dehennin L.Steroid 21-hydroxylase activity of the microsome-enriched fraction of follicular linings from equine ovaries has been demonstrated by gas chromatography-mass spectrometry. The 21-hydroxylated metabolites were quantified by isotope dilution with deuterated analogues. The two most abundant potential substrates for follicular steroid 21-hydroxylase, progesterone (P) and 17-hydroxyprogesterone (17OHP), were converted respectively to 11-deoxycorticosterone (DOC) and 11-deoxycortisol with corresponding apparent specific activities of 308 and 24 pmol/mg protein/h and apparent Km values of 1.1 and 6.4...
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...
You Y, Proctor RM, Vasilko ED, Robinson MA.JWH-250 is a synthetic cannabinoid. Its use is prohibited in equine sport according to the Association of Racing Commissioners International (ARCI) and the Fédération Équestre Internationale (FEI). A doping control method to confirm the presence of four JWH-250 metabolites (JWH-250 4-OH-pentyl, JWH-250 5-OH-pentyl, JWH-250 5-OH-indole, and JWH-250 N-pentanoic acid) in equine urine was developed and validated. Urine samples were treated with acetonitrile and evaporated to concentrate the analytes prior to the analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The chromat...
Francfort P, Schatzmann HJ.It is shown that the concentration of ouabain necessary for 50 per cent inhibition of the Na+K activated membrane ATPase of red cells is similar in man and horse. This is taken to indicate that the two species have similar sensitivity towards cardiac glycosides in general. In five adult healthy horses plasma digoxin concentration was measured with a radioimmunoassay technique after a single intravenous injection of 1 mg/100 kg body weight digoxin. The half time of elimination was 23 h and the apparent volume of distribution 7.3 litres/kg. An approximate estimate of plasma protein binding of di...
Wang J, Wen S, Zhao R, Qi J, Liu Z, Li W, An J, Wood C, Wang Y.The conjugation of small ubiquitin-like modifier (SUMO) to the target protein, namely, SUMOylation, is involved in the regulation of many important biological events including host-pathogen interaction. Some viruses have evolved to exploit the host SUMOylation machinery to modify their own protein. Retroviral Gag protein plays critical roles in the viral life cycle. The HIV-1 p6 and the Moloney murine leukemia virus CA have been reported to be conjugated with SUMO. In this study, we report for the first time, to our knowledge, the covalent conjugation of equine infectious anemia virus (EIAV) G...
Belford CJ, Lumsden JH.This report is an assessment of clinical chemistry dry reagent methodology for veterinary use. A portable reflectance photometer and dry reagent strips were used to measure canine whole blood hemoglobin, and total bilirubin, glucose, cholesterol, creatinine and urea in canine, bovine, equine and feline sera. Creatine kinase and lactate dehydrogenase were assayed in canine, bovine and equine sera. The following aspects of performance are reported: within run variation determined on canine samples, between run variation using a commercial control, correlations between dry reagent and wet reagent...
Frohlich M, Knights K, Springer NL.Differentiating immune-mediated causes from other causes of anemia and thrombocytopenia can be challenging. Flow cytometry can detect surface-associated immunoglobulin (sIg) on red blood cells (RBC) and platelets (PLT) in dogs and horses. Sample storage parameters for ideal assay performance has not been evaluated in horses. The study objective is to identify optimal storage time and temperature of equine whole blood for the detection of RBC-sIg and PLT-sIg via flow cytometry. Both assays were performed on samples at time 0, 4, 24, 48, and 72 h post collection. RBC-sIg samples were stored at ...
Sedgwick AD, Dawson J, Lees P.Subpopulations of equine leucocytes, polymorphonuclear and mononuclear cells, were separated from whole blood on a discontinuous Percoll gradient and used in studies of chemokinesis and chemotaxis. Polymorphonuclear cells responded to the chemo-attractant properties of zymosan-activated plasma in Boyden chamber and agarose microdroplet assays but they responded only slightly (Boyden chamber) or not at all (agarose microdroplet) to the peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). Equine mononuclear cell movement was increased by FMLP in both assay systems and these cells also respond...
Berryhill EH, Urbina NS, Marton S, Vernau W, Alonso FH.The Wellness Ready Test (WRT) is a lateral flow, stall-side assay that measures equine insulin in whole blood and requires validation before recommending clinical use. We evaluated intra- and inter-assay precision and linearity and compared the WRT with a radioimmunoassay (RIA). Tested concentrations ranged from 695 pmol/L (100 μIU/mL). For 20 replicates at each insulin level, intra-assay CVs of the WRT for insulin were 13.3%, 12.9%, and 15.3% at low (139-278 pmol/L; 20-40 μIU/mL), intermediate (278-417 pmol/L; 40-60 μIU/mL), and high (>417 >60 μIU/mL) concentrations...
Houghton E, Oxley GA, Moss MS, Evans S.A method is described to confirm the presence of 19-nortestosterone metabolites in urine after the administration of veterinary preparations of this anabolic steroid to horses. The method is based upon the detection, by gas chromatography mass spectrometry or selected ion monitoring, of an isomer of estrane-3,17-diol in the urine.
Bijman J, Quinton PM.The effects of Ca2+, the cyclic nucleotides adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP), and other parameters of sweat secretion from single equine sweat glands were examined in vitro. Extracellular Ca2+, the Ca2+ ionophore A23187, and the Ca2+ channel antagonist verapamil were all without effect on sweat secretion. Prolonged rinsing of the glands in Ca2+-free Ringer solution with 5 mM ethylenediaminetetraacetic acid decreased the secretion to 30% of the control sweat rate in response to the beta-adrenergic agonist isoproterenol; the sweat respon...
Kirkland PD.During the 2007 equine influenza (EI) outbreak in Australia, diagnostic laboratories and the use of appropriate tests played a pivotal role in the response to the crisis. This role began with the detection of EI virus in New South Wales (NSW) on the evening of 24 August 2007 and culminated in providing the final 'proof of freedom' from EI in March 2008. The tests that were used during the EI response were able to provide results quickly, and with high sensitivity and specificity. This section of the supplement describes the roles and functions of the Australian laboratories; tests used and the...
Hochi S, Fujimoto T, Oguri N.Viability following vitrification of equine blastocysts with different sizes was investigated in vitro. Twenty-four blastocysts were classified into three groups according to their diameters ( 300 microns; n = 8 each). The solution used for vitrification was defined as EFS and contained 40% ethylene glycol, 18% Ficoll and 0.3 M sucrose in modified-phosphate-buffered saline (m-PBS). During pretreatment with 20% ethylene glycol in m-PBS for 20 min, the larger blastocysts responded to the osmotic pressure caused by 20% ethylene glycol more slowly than the smaller blastocysts. Single blastocysts w...
