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.
Williams MA, Harrison PM.Horse ferritin was fractionated both by starch-gel electrophoresis and by gel filtration on Sephadex G-200. Monomer fractions contained up to 98% of monomer and oligomer fractions up to 76% of oligomers as determined by quantitative electron microscopy. Percentages obtained from electron micrographs correlated well with analytical starch-gel electrophoretograms and ultracentrifuge patterns. Amino acid analyses of monomer- and oligomer-enriched fractions showed no significant differences. Ferritin oligomers did not apparently dissociate on dilution for electron microscopy or on storage. Apoferr...
Turner GA, Taylor DM.The interactions between tetravalent plutonium and horse serum proteins were studied in vitro by electrophoresis on cellulose acetate and by gel filtration. The results show that in horse serum, as in other mammalian sera, the plutonium is associated principally with the transferrin component of the beta1-globulins. The formation of the plutonium-transferrin complex requires the presence of HCO3-, and plutonium is displaced from the complex by excess iron, thus indicating that similar binding sites may be involved in the complexing of iron and plutonium. The plutonium complex is considered to ...
Babel I, Stella RC, Prado ES.Previous experiments indicated that horse urinary kallikrein (UK) hydrolyzes salminei- e and polyarginine, a but not polylysine. This paper reports the action of UK on bradykinyl-serine, methionyllysyl-bradykinin and lysyllysyl-bradykinin.
Lee CL, Zandrew F, Davidsohn I.One hundred infectious mononucleosis and the same number of non-infectious mononucleosis sera were studied to evaluate the sensitivity and specificity of horse erythrocytes in the diagnosis of infectious mononucleosis. Titres of horse agglutinins in infectious mononucleosis sera ranged from 28 to 7,168 with a geometric mean of 550, whereas the corresponding sheep agglutinin titres ranged from less than 7 to 3,584, with a geometric mean of 126. Horse agglutinin titres of non-infectious mononucleosis sera ranged from less than 7 to 896, with a geometric mean of 59. Infectious mononucleosis sera ...
Karawya MS, El-Keiy MA, Wahba SK, Kozman AR.A chromatographic separation of amphetamine, methylamphetamine and ephedrine from horse urine is possible on alkaline Silica Gel G plates developed with acetone-methanol (1:3). After elution, the bases are determined colorimetrically. The intensity of the violet colour resulting from the nitration of amphetamine is measured in a Unicam SP1300 colorimeter using filter No. 1 (sensitivity 50–250 μg). The colour produced by the interaction of methylamphetamine, sodium nitro-prusside, acetaldehyde and triethanolamine is measured at 590 mμ (sensitivity 200–2,000 μg). Ephedrine was determined ...
O'Callaghan DJ, Hyde JM, Gentry GA, Randall CC.Infection of exponential-phase suspension cultures of mouse fibroblast cells (L-M) with equine abortion virus (EAV) resulted in inhibition of cell growth and marked alterations in host metabolic processes. The synthesis of deoxyribonucleic acid (DNA) and ribonucleic acid was inhibited within 4 hr after infection and was suppressed by more than 90% by the time of maximal virus replication (14 to 18 hr). The overall rate of protein synthesis, however, was similar in uninfected and virus-producing cells as determined by measurements of net protein and isotope incorporation. The time course of vir...
Kriegshäuser G, Kuechler E, Skern T.Equine rhinitis A virus (ERAV) is a picornavirus which causes an acute respiratory infection in horses worldwide, and virus neutralization (VN) has been the standard method for the detection of ERAV antibody in horse serum. Previous studies have identified recombinant virion protein VP1 (rVP1) purified under native conditions to be of high potential for the development of a diagnostic ERAV enzyme-linked immunosorbent assay (ELISA). This study presents an optimized protocol for the expression and purification of native full-length rVP1. Furthermore, we demonstrate that, upon denaturation, rVP1 ...
Dirikolu L, Lehner AF, Hughes C, Karpiesiuk W, Camargo FC, Harkins JD, Woods WE, Bosken JM, Boyles J, Troppmann A, Fisher M, Tobin T.Furosemide is a potent loop diuretic used for the prevention of exercise-induced pulmonary hemorrhage in horses. This drug may interfere with the detection of other substances by reducing urinary concentrations, so its use is strictly regulated. The regulation of furosemide in many racing jurisdictions is based on paired limits of urinary SG (100 ng/ml). To validate this regulatory mechanism, a liquid chromatography/mass spectrometry/mass spectrometry method employing a solid-phase extraction procedure and furosemide-d5 as an internal standard was developed. The method was used to determine th...
Brown L, Townsend W, Waltisbuhl D.The unique challenges that laboratories in Queensland and New South Wales faced during the response to the 2007 equine influenza outbreak and how these were managed are described.
Kearns CF, Lenhart JA, McKeever KH.Erythropoietin (EPO) is the primary hormone of erythropoiesis. Administration of recombinant human erythropoietin (rhuEPO) to improve racing performance in the horse represents a new form of blood doping, which has been associated with increased mortality. While immunoassay kits have become plentiful, very few commercial hormone assays are made specifically for equine research. There is a strong degree of sequence homology reported for EPO among species, which has allowed antibodies designed for human EPO research to be used to determine EPO concentration in other species. The objective of the...
Fukunaga Y, Wada R, Sugita S, Fujita Y, Nambo Y, Imagawa H, Kanemaru T, Kamada M, Komatsu N, Akashi H.Equine arteritis virus (EAV) was readily isolated in RK-13 cell monolayers by plaque assay from seminal plasma of experimental carrier stallions when they contained high titers of virus regardless of the presence of non-viral cytotoxicity in the seminal plasma. The cytotoxicity interfered with virus isolation from seminal plasma which contained virus at titers less than 10 PFU/ml. However, it was possible to detect the virus in seminal plasma pretreated with PEG (#6000). EAV was consistently identified by RT-PCR from crude seminal plasma which contained virus at titers of more than 10(2.7) PFU...
Jacobi S, Townsend WM, Bolin CA.To evaluate whether equine serum administered via a simulated subpalpebral lavage system (SPL) supports proliferation of Streptococcus zooepidemicus or Pseudomonas aeruginosa within the tubing. Methods: A sterile i.v. catheter with injection cap was inserted into sterilized silicone tubing (Mila). To mimic an SPL within the dorsal conjunctival fornix, the tubing was secured to an elevated platform. The tip of the tubing extended from the platform into a vial containing culture medium just inoculated with approximately 1.5 x 10(8) CFU/mL P. aeruginosa or S. zooepidemicus. To mimic administratio...
Söylemez Z, Ozer I.The inhibition of horse plasma cholinesterase by propranolol showed characteristics which depended upon the identity of the substrate used. With butyrylthiocholine as substrate, the inhibition showed a first-order dependence on inhibitor concentration, and was characterized by a Ki of 8 microM (pH 7.4, 20 degrees C). With p-nitrophenylbutyrate as substrate, a biphasic v-1 versus [I] relationship was obtained. The biphasic curve could be resolved into two components, with apparent Ki's of 9 microM and 1.3 mM. Use of butyrylthiocholine as alternative substrate resulted in partial inhibition of p...
Selwet M.The research concerned the determination of the frequency of occurrence of selected virulence genes (cadF, flaA, iam) and genes responsible for the formation of the CDT cytotoxin (cdtA, cdtB, cdtC) in Campylobacter spp. The research object consisted of 100 faecal samples collected from stallions showing no symptoms of campylobacteriosis. The presence of bacteria of the genus Campylobacter spp. Was found in 25 individuals (25%). The molecular biology techniques used in the research allowed us to distinguish the following species from the positive samples: C. jejuni (68%); C. coli (28%) and C. l...
Dai CB, Xiao Y, Lu H, Shen RX, Shao YM.Membrane protein GP90 of China equine infectious anemia virus (EIAV) vaccine strain (DLV) and its parental wild type LN strain were expressed with Bac-to-Bac baculovirus expression system and BALB/c mice were inoculated with purified protein, thereby to explore the availability of protein for differential diagnosis and potential for preparing genetically engineered vaccine. Methods: The authors infected donkey PBMC culture with China EIAV vaccine strain (DLV) and its parental wild type LN strain, extracted its proviral DNA as template, amplified the GP90 of DLV and LN, respectively, and expres...
Dimsoski P.To show that application of the polymerase chain reaction (PCR) method modified for amplification of a low-copy number DNA samples, ie, the isolation of PCR products (IPCRp), would represent improvement in obtaining genotypes from a fecal DNA compared with previously used genotyping methods. Methods: The DNA from the horse fecal matter was extracted by modified Qiagen DNA Stool Mini Kit protocol. Following the extraction, the DNA genotypes from fecal samples were obtained by the most powerful PCR amplification method, the IPCRp. The IPCRp-based multiplex kit amplified biotin-labeled strands we...
Grosskopf JF, Smuts EG.Blood was collected from 28 zebra mare (Equus burchellia antiquorum) immediately after being shot in the Kruger National Park. The serum was separated within two hours after collection and then stored at -15 degrees C for later assay. Of these, thirteen selected samples were tested for gonadotrophic activity. The stage of pregnancy was determined from a foetal growth curve. Blood samples from pregnant horse mares were collected by venipuncture. Nine mares were sampled. Seven blood samples at different stages of pregnancy were collected from one mare, four from another and only one sample each ...
Sticht H, Willbold D, Rösch P.Two molecular dynamics (MD) simulations were performed in order to increase the understanding of the dependence of protein conformation on solvent environment. The protein used for these simulations is the transcriptional activator of the equine infectious anemia virus (EIAV-Tat). The structure of this protein has been determined by nuclear magnetic resonance (NMR) in aqueous solution (Willbold et al., Science 264, 1584 (1994)) and in 40% (v/v) trifluoroethanol (TFE) (Sticht et al., Eur. J. Biochem., submitted) showing considerable differences in the stability of the secondary structure elemen...
Galey FD.In most competent veterinary diagnostic laboratories, analytical findings are interpreted by the veterinary toxicologist to determine the significance of the finding in view of historic, clinical, and pathologic findings. A veterinary toxicologist also will provide consultation about possible toxic rule-outs for a case, treatment of affected animals, and prevention of additional cases. Once all of the information is available, a complete summary of the findings can be provided to the client. When the procedures outlined are followed, including a systematic approach to collecting all the eviden...
Neely M, Arroyo L, Jardine C, Clow K, Moore A, Hazlett M, Weese JS.The blacklegged tick (), which transmits , the causative agent of Lyme disease, has undergone rapid range expansion in Ontario. In horses, Lyme disease remains an enigmatic disease, with limited understanding of the pathogenesis and many issues pertaining to selection and interpretation of laboratory tests. We evaluated seropositivity in naturally exposed horses over a 12-mo period and compared paired samples with 2 common serologic tests. Serum samples were collected in 2017, ~1 y after initial testing, from a cohort of 22 horses that were seropositive in a 2016 seroprevalence study. Sampl...
Wilkołek P, Szczepanik M, Sitkowski W, Rodzik B, Pluta M, Taszkun I, Gołyński M.Although intradermal testing (IDT) is commonly used in the etiological diagnosis of allergies, in vitro testing for specific IgE (sIgE) is an attractive alternative. Currently, new laboratory techniques in veterinary allergological practice, including multiple allergen simultaneous tests (MASTs), gradually supersede in vivo tests. Both, serological (sIgE) and IDTs in fourteen atopic Malopolski horses were performed. Correlation and agreement between test results were evaluated. Receiver operating characteristic analysis showed that sIgE to had the best diagnostic performance (Area under the R...
Viu J, Armengou L, Ríos J, Muñoz A, Jose-Cunilleras E.To determine the strong ion difference (SIDa ) and total nonvolatile weak buffers (ATOT ) in healthy foals during the first year of life and to compare reference biochemistry laboratory with analyzers available during emergency hours. Methods: Prospective study performed over 2 years. Methods: University teaching hospital. Methods: Two hundred thirty-six healthy foals distributed in 6 groups: A (21 days-2 months), B (2-3 months), C (3-6 months), D (6-9 months), E (9-12 months), and 33 neonatal foals (< 21 days old). Methods: Blood samples were obtained to determine L-lactate, sodium, potass...
Paciello O, Pasolini MP, Navas L, Russo V, Papparella S.Central core disease is a nonprogressive or slowly progressive congenital myopathy with a variable degree of hypotonia and axial and proximal muscle weakness that is histologically characterized by areas devoid of oxidative enzyme activity, resulting from an absence or low numbers of mitochondria in these regions (central core). A 10-month-old, male, pony foal was examined because of stiff gait, marked contractures of the distal portion of the limbs, flexion deformities of the hooves, and moderate hypotonia that had been present from birth. The foal had increased creatine kinase (282 U/liter; ...
Luo Y, Rudy JA, Uboh CE, Soma LR, Guan F, Enright JM, Tsang DS.The method describes quantification and confirmation of flunixin in equine plasma by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC/Q-TOF/MS/MS). Samples were screened by enzyme-linked immunosorbent assay (ELISA) and only those samples presumptively declared positive were subjected to quantification and confirmation for the presence of flunixin by this method. The method is also readily adaptable to instrumental screening for the analyte. Flunixin was recovered from plasma by liquid-liquid extraction (LLE). The sample was diluted with 2 ml saturated phosphate buffer (pH ...
Chuang MS, Huang HH, Dixon KM, Chen KS, Mao CL, Chen CL.The pharmacokinetics of clenbuterol in equine urine and blood was investigated. Methods: Urine and blood samples were collected following 3-day multiple oral administrations. The samples were examined using enzyme-linked immunosorbent assay and further confirmed by solid phase extraction and capillary electrophoresis. Results: Urinary clenbuterol was detectable until day 14 after the last dose. The urinary excretion of clenbuterol was characterized by a biphasic pattern. The half-lives of the bi-exponential elimination (t(1/2alpha) and t(1/2beta)) for urinary clenbuterol were about 12.1 and 48...
Kim JY, Choi MH, Kim SJ, Chung BC.A high-performance liquid chromatographic-tandem mass spectrometric (HPLC/MS/MS) method for the determination of 19-nortestosterone and its esters (cyclopentanepropionate, phenylpropionate, and decanoate) in equine plasma is achieved using an atmospheric pressure chemical ionization (APCI) interface in selected reaction monitoring (SRM) mode. The two internal standards used were 16,16, 17-(2)H(3)-19-nortestosterone for 19-nortestosterone and methenolone acetate for its esters. The steroids studied were extracted from plasma samples with a mixture of diethyl ether/n-hexane (9:1, v/v). The quant...
Stanley S, Jeganathan A, Wood T, Henry P, Turner S, Woods WE, Green M, Tai HH, Watt D, Blake J.We have raised antibodies to morphine and etorphine and developed one-step enzyme-linked immunosorbent assays (ELISA) for these drugs as part of a panel of post race tests for drugs in racing horses. These tests are simple, can be completed in 2 h, and can be read by visual inspection. The morphine ELISA has an I50 for morphine of about 1.5 ng/mL, while the etorphine ELISA has an I50 for etorphine of 250 pg/mL. Cross-reactivity studies show that the antimorphine antibody cross-reacts well with levorphanol, hydromorphone, and oxycodone, while the anti-etorphine antibody showed no cross-reactivi...
Bertram CA, Dietert K, Pieper L, Erickson NA, Barton AK, Klopfleisch R.During bronchoalveolar lavage fluid (BALF) sample preparation in horses, several technical aspects can affect sample variability. To date, the effects of different fixatives on prepared equine BALF films have been insufficiently investigated. Objective: This study aimed to determine the effect of various on-slide fixation methods on cell quality, including spray fixation of wet films, and acetone and methanol fixation of air-dried samples in comparison with unfixed, air-dried films. Methods: Cytocentrifuged BALF samples from 5 horses were fixed in a wet state using a commercially available fix...
Decloedt A, Bailly-Chouriberry L, Vanden Bussche J, Garcia P, Popot MA, Bonnaire Y, Vanhaecke L.Doping control is a main priority for regulatory bodies of both the horse racing industry and the equestrian sports. Urine and blood samples are screened for the presence of hundreds of forbidden substances including anabolic-androgenic steroids (AASs). Based on the suspected endogenous origin of some AASs, with β-boldenone as the most illicit candidate, this study aimed to improve the knowledge of the naturally present AAS in horse urine. To this extent, a novel ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated according to...
Hagedorn HW, Meiser H, Zankl H, Schulz R.Due to its tranquilizing properties, the tricyclic antidepressant doxepin may be misused as a doping agent in competition horses. Therefore, efficient analytical procedures are required to detect this drug in samples submitted for doping control. To screen for parent doxepin in equine blood and urine, a less specific method has been accepted employing gas chromatography (GC) combined with electron impact (EI) mass spectrometry (MS). The aim of this study was identification of doxepin metabolites providing more specific MS data to verify positives resulting from screening. Thus, after a horse w...
de Vries PJ, van der Holst W.The reliability of determination of the plasma progesterone level within approximately eighteen days after ovulation in the pregnancy diagnosis of mares is examined in the present study. Studies were done in seventy-five mares, a number of which were served or inseminated during several cycles so that a total number of eighty-seven blood samples were obtained. On the analogy of other authors, the progesterone level above which mares were believed to be pregnant and below which they were assumed to be non-pregnant, was set at 2 ng/ml. The twenty-five mares in which the level was below 2 ng/ml. ...
Yang JM, Woods WE, Weckman TJ, Wood TW, Chang SL, Blake JW, Tobin T.1. Drug administration studies using diisopropylamine dichloroacetate (DADA) and diisopropylamine (DIPA) were conducted in Thoroughbred and Standardbred horses to assess physiological effects and develop detection methods. 2. Four horses received 0.08 mg DADA/kg body wt and showed no changes in heart and respiratory rates or body temperature as measured over a 1-hr period after administration. A transient diuretic effect was found to occur in 2 mares dosed with 0.80 mg DADA/kg body wt. 3. A qualitative detection method using thin-layer chromatography was developed to detect DIPA, the major met...
Milenkovic D, Mata X, Chadi S, Guérin G.Laminins are large heterotrimeric basement membrane glycoproteins composed of alpha, beta and gamma chains. The Laminin 5 isoform has an alpha3beta3gamma2 composition and is essential for the adhesion of basal keratinocytes to the underlying epithelial basement membrane where it is mainly located. Mutations in the genes coding for the 3 chains have been associated with a severe skin blistering disease, Herlitz's junctional epidermolysis bullosa (JEB), observed in different species as man, dog, cat and horse. In this study, we report the sequence of the 5.2 kb horse laminin alpha 3 cDNA (LAMA3)...
Moeller BC, Clifford A, Emery RT, Alarcio G, Favro G, Arthur RM.Zilpaterol is a β -adrenergic agonist and a repartitioning agent that has a high potential for abuse in equine performance athletes. Analysis of zilpaterol in hair is an alternative sampling matrix that extends detection time periods beyond those found in urine or blood samples. Our laboratory has been screening for zilpaterol in hair for many years and recently detected and confirmed its presence in official samples. Accordingly, a liquid chromatography-mass spectrometry method was developed and validated to detect and confirm zilpaterol in equine hair. Briefly, equine hair was decontaminate...
Kuemmerle JM, Kühn K, Bryner M, Fürst AE.To evaluate if the use of locking head screws (LHS) in the distal holes of a locking compression plate (LCP) applied to the caudal aspect of the ulna to treat equine ulnar fractures is associated with a risk of injury to the lateral cortex of the radius. Methods: Controlled laboratory study. Methods: Cadaveric equine forelimbs (n = 8 pair). Methods: After transverse ulnar osteotomy, osteosynthesis was performed with a narrow 10-13 hole 4.5/5.0 LCP applied to the caudal aspect of each ulna. The distal 3 holes were filled with 4.5 mm cortex screws (CS) in 1 limb (group 1) and with 5.0 mm LHS con...
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...
