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.
Baldwin DM, Roser JF, Muyan M, Lasley B, Dybdal N.Enzymatically dispersed anterior pituitary cells from donor mares were cultured for 48 h in alpha-modified Eagles' medium containing 10% steroid-free horse serum. The cells were then incubated for 24 h in fresh medium oestrogen followed by a 4-h incubation with or without GnRH. Media and cell extracts were analyzed for LH by radioimmunoassay. In the first series of experiments, pituitary cells from Day-3 dioestrous mares were preincubated with ethanol (control) or different concentrations of E2 (10(-11) to 10(-7) M) for 24 h prior to a 4-h incubation without (basal) or with 1.0 nM GnRH. E2 inc...
Tucker KE, Henderson KA, Duby RT.Twenty-three follicles were collected from 14 mares on specific days and grouped to represent follicles from early (Group 1; n = 6), mid (Group 2; n = 11) and late (Group 3; n = 6) oestrus, as described previously (Tucker et al., 1988). Isolated granulosa cells (GC) from each follicle were cultured in multiwell plates containing either Eagle's Minimum Essential Medium (MEM) alone, eLH (300 ng/ml), eFSH (300 ng/ml) or eLH + eFSH (300 ng/ml each), in the presence or absence of 0.5 microM testosterone. Media were collected and replaced at 24 h of culture, and 24 h later, media were again collecte...
Andrianov AM, Akhrem AA.Using the earlier suggested method the calculation of the backbone conformations of horse heart cytochrome c in oxidized (ferricytochrome c) and reduced (ferrocytochrome c) states has been performed by the two-dimensional nuclear Overhauser effect spectroscopy data. For both protein forms the secondary structure elements have been revealed and the conformations of the irregular polypeptide chain segments have been analysed. The similarity of the secondary structures of ferri- and ferrocytochrome c in solution was established from the comparison of their conformations. Small differences between...
Houghton E, Holtan D, Grainger L, Voller BE, Rossdale PD, Ousey JC.Radioimmunoassay (RIA) and gas chromatography-mass spectrometry (GC-MS) were used to determine plasma progestagen concentrations in the normal and premature foal. Radioimmunoassay provides a profile of plasma progestagens with respect to time but, due to the non-specific nature of the technique and without prior chromatographic purification, quantitative data based on RIA analysis must be interpreted with caution. In contrast, the greater specificity of GC-MS allows identification of specific plasma progestagens and measuring of multiple analytes in a single analysis. Both techniques demonstra...
Hierholzer JC, Stone YO, Broderson JR.Reference equine antisera to all 47 serotypes of human adenoviruses presently described have been prepared and evaluated by reciprocal neutralization and hemagglutination-inhibition tests. All tests were carried to endpoint dilutions a minimum of five times in each direction to give accurate values for homologous and heterologous antibody titers. Significant cross-reactions in the horse antisera were compared to similar data obtained from rabbit antisera. Using this analysis, major antigenic relationships exist among types 12-18-31 of subgenus A, types 7-11-14 and 34-35 of subgenus B, types 8-...
Angelos JA, Marti E, Lazary S, Carmichael LE.The DNA from equine sarcoid samples from New York State and Switzerland was isolated and probed with bovine papillomavirus type 1 (BPV-1) to determine if BPV genomes were present. Twelve of 13 sarcoids from New York State and 17/20 sarcoids from Switzerland contained DNA that hybridized to the BPV-1 probe. Restriction enzyme analysis of the positive samples demonstrated restriction fragment profiles characteristic of BPV-1 in 22 sarcoids and restriction fragment profiles characteristic of bovine papillomavirus type 2 (BPV-2) in 7 sarcoids. In addition, three tissues histologically diagnosed as...
Nielsen AH, Gotfredsen P, Nielsen PB, Hyttel P, Poulsen K.1. Species specific problems complicating the measurement of prorenin and renin concentrations were studied in bovine, hog and horse plasma. 2. In contrast to horse renin, bovine and hog renin reacted with rat angiotensinogen, allowing measurement of the plasma renin concentration in cattle and hog with rat angiotensinogen as exogenous substrate. 3. Trypsin treatment of plasma in order to activate prorenin generated an interfering angiotensin I immunoreactive material in all three species, most extensively in horse plasma. 4. This material could be removed in bovine and hog plasma by a cation-...
Pellegrini A, Waiblinger S, Von Fellenberg R.Lysozyme from equine neutrophil granulocytes was isolated in a pure form by fast performance liquid chromatography, i.e. ion-exchange chromatography and reversed-phase chromatography. The lysozyme lysed Micrococcus luteus, Bacillus subtilis and Staphylococcus lentus and was also bactericidal against the Gram-negative bacteria Escherichia coli, Klebsiella pneumoniae, Bordetella bronchiseptica, and Serratia marcescens. Staphylococcus aureus and Staphylococcus epidermidis were not lysed. The lysozyme was only very slightly bactericidal for S. epidermidis and S. aureus. Equine neutrophil lysozyme ...
Baker CB, Adams MH, McDowell KJ.Horse conceptuses were collected on Days 13, 15, 20 and 25 after ovulation. Whole conceptuses (Days 13 and 15) or extra-embryonic membranes (Days 20 and 25) were homogenized and poly-adenylated RNA (poly A RNA) was isolated by binding to oligo (dT)-cellulose. Poly A RNA (1 microgram/well) was separated by size on a denaturing 1% agarose gel and blotted onto nitrocellulose filters (northern blotting). DNA probes were prepared from plasmids containing equine alpha 1, omega 1 and omega 2 interferons and human beta actin. The presence of messenger RNA (mRNA) was detected by specific hybridization ...
Boermans HJ, Johnstone I, Black WD, Murphy M.Six horses gavaged with a commercial brodifacoum (BDF)-containing bait (Talone) at a dosage of 0.125 mg of BDF/kg of body weight showed weight loss, severe hypocoagulability and hemogram alterations. Four of the horses became depressed and anorectic; one required vitamin K1 therapy. Increases in clotting times were observed at 24 h in the partial thromboplastin time (PTT) followed by the thrombotest (TBT) and one-stage prothrombin time (PT) at 48 h. Elevated mean PTT, PT and TBT were observed from days 4 to 8 (p less than 0.05) with levels returning to pretreatment levels by day 12. Maximum pr...
Hermanson JW, Hegemann-Monachelli MT, Daaod MJ, LaFramboise WA.The biceps brachii of horses is subdivided into a lateral and medial head. Electrophoresis of samples from the lateral head revealed three slow-migrating native myosin isoforms, including one that does not correspond to slow myosin isoforms described for other mammalian muscles. In contrast, the medial head contained a single slow isoform. Both the lateral and medial heads contained three fast-migrating isoforms corresponding with the FM-2, FM-3 and FM-4 isoforms reported for other mammalian fast-twitch muscle fibers. Electrophoresis of myosin heavy chains (MHCs) revealed only two MHC bands, o...
McFarlane JR, Cabrera CM, Coulson SA, Papkoff H.The rhinoceros is an endangered species related to the horse family. Little is known of its reproductive endocrinology. The objectives of this study were to partially purify rhinoceros pituitary hormones, determine which assays could be used for their assessment, and to ascertain whether rhinoceros LH possesses the intrinsic FSH activity of equine LH. A single pituitary each from a White (1.3 g) and a Black (1.2 g) Rhinoceros was homogenized and extracted (pH 9.5), then subjected to pH and salt fractionation, and ion-exchange chromatography (DEAE and Sephadex SP-C50) to yield partially purifie...
Baxter GM, Moore JN, Tackett RL.The in vitro responses of isolated vascular preparations of digital arteries and veins obtained from healthy anaesthetised horses were determined for dopamine and fenoldopam. The digital vessels were harvested, cut into 4 mm vascular segments, suspended in tissue baths and attached to force-displacement transducers. Dose-response studies between 10(-8) and 10(-4)M concentrations were performed for all drugs. The change in tension of each vascular ring was measured in grams of force. The reactivity between palmar and plantar digital vessels and baseline vascular responses were determined for do...
Ledwozyw A, Kadziołka A, Jabłonka S, Tusińska E, Herbut M.The aim of this study was to investigate the platelet factor 3 activity in platelet-rich plasma of horse, cattle, sheep and pig, by the use of chromogenic tripeptide substrate H-D-Phenylalanyl-Pipecolyl-Arginyl-p-nitroanilide. Among species examined the highest activity of this factor was stated in pig, the lowest one in sheep. Chromogenic substrate test was 10 times more sensitive that Stypven clotting time test. Thus, the use of chromogenic tripeptide substrate is fully valuable in platelet factor 3 activity estimation in domestic animals.
Fayrer-Hosken RA, Caudle AB, Shur BD.beta 1, 4-Galactosyltransferase (GalTase) is localized to the plasma membrane of mouse sperm, in which it mediates the binding of sperm to glycoconjugate residues in the egg zona pellucida. In this study, the presence of subcellular distribution of sperm GalTase were determined in two other mammalian species that yield sufficient sperm for subcellular fractionation. Equine and bovine semen were collected, and the plasma membranes (PM), outer acrosomal membranes (OAM), and inner acrosomal membranes (IAM) were sequentially removed. The purities of the isolated membrane preparations were determin...
Cocco MJ, Lecomte JT.A proton nuclear magnetic resonance spectroscopic study of horse apomyoglobin was undertaken in order to define the regions of myoglobin that are and that are not structurally affected by the binding of the prosthetic group. It was found that, in spite of the poor spectral resolution, a number of spin systems could be identified by using standard correlated methods. Four clusters consisting mostly of hydrophobic residues were detected by nuclear Overhauser spectroscopy, two of which involved the tryptophan side chains. Extensive similarities to nuclear Overhauser spectroscopy data collected on...
Kadir FH, Moore GR.Horse spleen ferritin, a spherical protein shell of 24 subunits, contains no haem when extracted. This contrasts with ferritins isolated from bacterial sources which have the capacity to bind up to 24 haem groups [(1990) FEBS Lett. 271, 141-143] via two methionine residues [(1990) Nature 341, 771]. Here it is shown that horse spleen ferritin can bind between 15 and 17 haems per 24 subunits with an apparent association constant of 2.2-3.2 x 10(4) M-1. The strength of haem binding appears to be unaffected either by the presence of the core or by the oxidation state of the haem. The demonstration...
Evers P, Hoffmann B.This paper describes the use of hormone analysis in the diagnosis of adrenal cortex dysfunction in the dog, cat and horse. Analytical problems concerning the determination of corticosteroid levels are discussed and the pathology of adrenal dysfunction is briefly presented. The paper focuses on the problems in establishing physiological norms for adrenal function based on the established assays. Own experiences and other reported data are referred to.
Van Heerden J, Dauth J, Dreyer MJ, Nichas E, Marshall C, De Waal DT.Selected haematological, blood chemical and serological variables were investigated in healthy Thoroughbreds (n = 45) in training. Haemoglobin concentration, haematocrit, red, white and differential cell counts as well as serum concentrations of total and ionized calcium, sodium, potassium, chloride, urea, creatinine, total protein, albumin, inorganic phosphorus, total bilirubin, iron, glucose, magnesium, alkaline phosphatase, gamma-glutamyltransferase, lactate dehydrogenase, aspartate transaminase, alanine transaminase and creatine kinase were found to be within ranges previously reported for...
Vachon AM, Keeley FW, McIlwraith CW, Chapman P.Articular cartilage specimens from the distal articular surface of 32 radiocarpal bones from 24 2- to 5-year-old horses were analyzed. The total collagen content was determined on the basis of the 4-hydroxyproline content, using a colorimetric method. A method for estimating the proportions of types-I and -II collagen by measuring spectrophotometric densities of specific cyanogen bromide peptide bands from mixtures of types-I and -II collagen on sodium dodecyl sulfate-polyacrylamide gels was used. The cyanogen bromide peptides representative of each collagen types-I and -II were identified. Th...
Saman E, Breugelmans K, Heyndrickx L, Merregaert J.The genome of equine infectious anemia virus (EIAV) contains several small open reading frames (ORFs), the importance of which in the development of the virus is not clear. We investigated the possibility that the largest of these ORFs (ORF S3) is expressed during the course of the viral infection. The ORF S3 information was expressed in Escherichia coli, and the antigen was used to raise monospecific antiserum. A 20-kDa protein expressed in cells producing EIAV was identified as the gene product of ORF S3. Furthermore, sera from EIAV-infected animals specifically recognized this protein, indi...
Pemberton AD, Hodgson JC, Gilmour JS, Doxey DL.Isolates were prepared from the sera of 12 horses with acute grass sickness, using methods reported to yield serum fractions associated with neurotoxicity, and their components identified by liquid chromatography and spectroscopy. All isolates were found to contain cortisol and six isolates also contained a degradation product of an analgesic drug, dipyrone. However, no recognised neurotoxin was detected.
Monteiro-Riviere NA, Bristol DG, Manning TO, Rogers RA, Riviere JE.Studies in dermatology, cutaneous pharmacology, and toxicology utilize skin from different animal species and body sites. However, regional differences exist in topical chemical percutaneous absorption studies in man and in animal. The objective of this study was to compare epidermal thickness and number of cell layers across species and body sites using both formalin-fixed paraffin and frozen sections. Cutaneous blood flow determined by laser Doppler velocimetry (LDV) was compared to histologic data. Six animals of each of the following species were used: monkeys, pigs, dogs, cats, cows, hors...
Ekfalck A, Rodriguez-Martinez H, Obel N.Explants from the matrix of the stratum medium of the wall of the equine and bovine hoof each were cultured on a microporous membrane, using a standard culture medium. After incubation at 37 C, the outgrowth was a mixture of keratinocytes and fibroblasts, with predominance of the latter. After incubation at 34 C, the keratinocytes dominated, covering the lateral surfaces of the explant as well as the basal surface. Lateral outgrowth of keratinocytes was observed at the borderline of the original epidermis and at the borderline of the explant's contact with the membrane. Epithelial outgrowth fr...
Pereira A, Braekman JC, Dumont JE, Boeynaems JM.The incorporation of iodide into proteins (PBI) and lipids (LBI) of horse thyroid slices was measured in various conditions. Their dependency on the concentration of extracellular iodide was strikingly different. For PBI the relationship was biphasic with a decrease above 10 microM, likely to correspond to the Wolff-Chaikoff effect. On the contrary, LBI increased as a function of iodide concentration up to 100 microM. Methimazole (MMI) inhibited the incorporation of iodide into both LBI and PBI, but higher concentrations of MMI were required to depress LBI as compared to PBI. The inhibition of...
Ross PF, Nelson PE, Richard JL, Osweiler GD, Rice LG, Plattner RD, Wilson TM.Fumonisin B1 (FB1) and FB2 were isolated from corn cultures of both Fusarium moniliforme and Fusarium proliferatum. Respective concentrations in culture materials of FB1 and FB2 ranged from 960 to 2,350 and 120 to 320 micrograms/g for F. moniliforme and from 1,670 to 2,790 and 150 to 320 micrograms/g for F. proliferatum. Thin-layer chromatography, gas chromatography-mass spectroscopy, high-performance liquid chromatography, and liquid secondary ion mass spectroscopy were used for detection. Fumonisins from F. proliferatum have not previously been reported.
Kawamura S, Hirayama K, Mitsuoka T.Two chromogenic Limulus amebocyte lysate systems were evaluated for the measurement of endotoxic activity in feces of normal horses. Fecal extracts had neither non-specific nor inhibitory effect on the test reaction, and the two systems with different specificity showed equivalent results. Endotoxic activities in feces of healthy horses averaged 5.7 +/- 2.8 microns/g wet weight. Correlation between the endotoxin levels and the fecal microbial flora could not be determined.
Hinrichs K, Schmidt AL, Memon MA, Selgrath JP, Ebert KM.Embryos were recovered from the uteri of mares 5 d after ovulation. Six embryos, all morulae, were placed singly in 200-ul droplets of Ham's F-12 with 10% fetal calf serum and cultured at 37 degrees C in a 5% CO(2) atmosphere. The embryos expanded to form blastocysts by the third day of culture. The blastocysts hatched from their zona pellucida, rather than the zona thinning and flaking off, as occurs in vivo. Hatching from the zona pellucida began on the third day of culture and was complete in five of six embryos by the sixth day. The embryonic capsule, normally present in equine embryos aft...
Zhang J, Boyle MS, Smith CA, Moore HD.The acrosome of the stallion spermatozoon was visualized by indirect immunofluorescence with monoclonal antibody (18.6) which recognized an integral acrosomal membrane component. Localization was confirmed by electron microscopy using peroxidase labelled antibody. In fresh semen samples (n = 19), 73.9 +/- 9.1% of the spermatozoa from five fertile stallions displayed a uniform bright fluorescence over their acrosome region. In two semen samples from an infertile stallion only 28% and 35% of spermatozoa showed the same pattern of fluorescence. Spermatozoa from fertile stallions incubated for up ...
Lorenzo PL, Liu IK, Illera JC, Picazo RA, Carneiro GF, Illera MJ, Conley AJ, Enders AC, Illera M.Epidermal growth factor (EGF) has been reported to promote different functions in mammalian ovaries, including oocyte maturation. The aim of the present study was to establish: that EGF influences oocyte maturation in ovine and equine, that a tyrosine kinase-dependent intracellular mechanism mediates EGF effect and, that EGF-R receptor is detectable in ovarian follicles by immunohistochemistry methods. Selected ovine and equine oocytes were aspirated from 2-5 mm (ovine) or 25 mm (equine) follicles and cultured in TCM 199 for 22 (ovine) or 36 hours (equine). They are then subjected to culture w...
Recio P, Prieto D, MartÃnez MP, GarcÃa P, Rivera L, Benedito S, MartÃnez AC, Sacristán AG, Orensanz LM, Hernández M.Our aim was to study the presence of noradrenergic nerves and to characterize the alpha-adrenergic receptors involved in the contractions to electrical field stimulation and to alpha-adrenergic agonists of the horse penile deep dorsal vein. Noradrenergic fibres were visualized by immunohistochemistry using an antibody against dopamine-beta-hydroxylase (DBH). For functional studies, the responses of the venous rings to electrical field stimulation and to alpha-adrenergic agonists (noradrenaline, phenylephrine and BHT 920) were studied in the absence and the presence of noradrenergic transmissio...
Iametti BS, Tedeschi G, Oungre E, Bonomi F.In this work the purification and the complete primary structure of kappa-casein from equine milk are reported for the first time. Mares' milk casein was separated by RP-HPLC into four fractions. Complete primary sequence was obtained by sequence analysis of the protein in the fastest eluting peak isolated by chromatography. This sequence was 95% identical to that reported for the C-terminal portion of the zebras' kappa-casein and showed high similarity with kappa-caseins from sources other than Equidae, confirming that this protein was indeed kappa-casein in equine milk. The presence of post-...
Nakken G, Kirk J, Fjordbakk CT.Autologous conditioned serum (ACS), i.e serum enriched with anti-inflammatory cytokines and growth factors, is a popular orthobiologic therapy used in equine practice. Costly specialized tubes containing glass beads are commonly used for ACS production. The objective of this in vitro study was to compare cytokine and growth factor levels in equine serum after incubation in three different tubes: commercial plastic ACS tubes (COMM); sterile 50Â ml plastic centrifugation tubes (CEN); and 10Â ml plastic vacutainer tubes (VAC). Blood from 15 healthy horses was incubated in the different tubes at 3...
Tozaki T, Kikuchi M, Kakoi H, Hirota KI, Mukai K, Aida H, Nakamura S, Nagata SI.Transcriptome analyses based on DNA microarray technology have been used to investigate gene expression profiles in horses. In this study, we aimed to identify exercise-induced changes in the expression profiles of genes in the peripheral blood of Thoroughbred horses using DNA microarray technology (15,429 genes on 43,603 probes). Blood samples from the jugular vein were collected from six horses before and 1 min, 4 hr, and 24 hr after all-out running on a treadmill. After the normalization of microarray data, a total of 26,830 probes were clustered into four groups and 11 subgroups showing si...
Kumar S, Malhotra DV, Dhar S.The present study aimed to develop Dot-ELISA, complement fixation test (CFT) and capillary tube agglutination test (CAT) for serodiagnosis of Babesia equi infection and to compare their sensitivity with each other. For this study, sequential serum samples were collected from four donkeys experimentally infected with B. equi up to 90 days post infection (P.I.). B. equi antigen was prepared from the blood of a donkey showing more than 80% parasitaemia. Dot-ELISA, CF and CA tests were standardized as per the standard method. While performing CFT, it was observed that CFT standardized for the donk...
Bogdan JR, Morley PS, Townsend HG, Haines DM.This study has tested the effect of using homologous or heterologous equine influenza A virus isolates to evaluate serum antibody levels to influenza A virus in vaccinated and naturally-infected horses. In addition, the potential effect of antigenic selection of virus variants in egg versus tissue culture propagation systems was studied. Serum antibody levels in samples from horses recently infected with a local influenza A virus isolate (A/equine 2/Saskatoon/1/90) or recently vaccinated with a prototype isolate (A/equine 2/Miami/1/63) were assessed by hemagglutination inhibition and by single...
Fletcher MA, Caldwell KE, Saez L, Latif Z.A sialoglycoprotein from horse erythrocytes was isolated in essentially homogeneous form and found to contain the neuraminidase-sensitive determinant of the horse erythrocyte for Paul-Bunnell heterophile antibodies of infectious mononucleosis. This reactivity was retained after covalent coupling of the antigen to latex particles. The latex reagent has greater stability (greater than 3 years) than either fresh or preserved horse erythrocytes. It can be used in a direct slide test; no absorption of the serum is necessary. The new test compared favorably with some standard tests for infectious mo...
Mund SJK, Corbett C, MacPhee DJ, Campbell J, Honaramooz A, Wobeser B, Barber SM.Horses often sustain cutaneous wounds and healing can be prolonged and difficult to treat. Compared to body wounds, limb wounds heal slower and are more likely to develop exuberant granulation tissue. Differences in healing rates and exuberant granulation tissue formation is attributed to abnormal cytokine profiles. CXCL8 and its receptor CXCR2 are involved in acute inflammation whereas CXCL10 and its receptor CXCR3 are involved in inflammation resolution. β- arrestin-2 regulates inflammation through internalization of G-protein coupled receptors (GPCRs) including CXCR2 and CXCR3. Gene expres...
Palma F, Ligi F, Soverchia C.This work is a comparative study of Na(+)-K+ and Ca(2+)-Mg2+ ATPase associated with the erythrocyte plasma membranes in different mammals. The method used to test the activity of these enzymes is based on quantitative measurements of ADP released during the reaction with HPLC: the chromatographic type is an Ion-Pair Reversed Phase. We have found that the levels of Ca2+ stimulated ATPase are higher than those of Na(+)-K+ ATPase in red blood cells of all the different mammalian species, with the only exception being lamb erythrocytes where the values of both the ATPase activities are almost equa...
Philip M, Karakka Kal AK, Subhahar MB, Karatt TK, Mathew B, Perwad Z, Graiban FM, Caveney MR, Sayed R, Kadry AM.According to previous research, aminorex is the major metabolite of levamisole; however, in the screening of levamisole-positive racehorse urine and plasma samples, aminorex could only be detected in trace amounts or not at all. In forensic laboratories, hydroxy levamisole and its phase II conjugates make it easier to confirm levamisole misuse and to differentiate between the abuse of levamisole and aminorex. This study aimed to identify the major levamisole metabolites that can be detected along with the parent drug. Methods: The study describes levamisole and its metabolites in thoroughbred ...
Riemersma DJ, Lammertink JL.A calibration method is presented by which the signals of mercury-in-silastic strain gauges (MISS), implanted in the tendons of in vitro loaded equine hindlegs, were converted to tendon loads. The relationships between MISS-signals and tendon loads were obtained from tensile-force tests applied to the tendons. Special attention was paid to the correction of the MISS-signals for amplitude-shifts resulting from internal repositioning of the MISS after tendon isolation and temperature differences. Shift corrections equivalent to tendon strains up to 2.8% were necessary in the in vitro experiment....
Chen JW, Uboh CE, Soma LR, Li X, Guan F, You Y, Liu Y.A novel multiplex of independent dinucleotide tandem repeat (DTR) loci was previously described that is capable of not only discriminating human and equine DNA, but of identifying a single equine source. We report a case in which a bloodstained syringe and two needles were found during inspection of a barn by inspectors of the Pennsylvania Racing Commissions. Using the multiplex and single-locus detection, all 21 equine DTR markers were detected in a suspect horse and two evidence samples, indicating the evidence samples came from the suspect animal. Only six markers were detected in the third...
Mori M, Ichibangase T, Yamashita S, Kijima-Suda I, Kawahara M, Imai K.In the doping tests currently used in horse racing, prohibited substances or their metabolites are usually directly detected in urine or blood samples. However, despite their lasting pharmaceutical effects, some prohibited substances are rapidly eliminated from horse urine and blood, making them difficult to detect. Therefore, new indirect biomarkers for doping, such as plasma proteins that are increased by the prohibited substances, have recently attracted much attention. Here, a fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS) method was adopted for hor...
O'Brien MA, Holmes MA, Duffus WP.Anti-tetanus toxoid (TT) antibody (Ig) levels in the supernatant of cultured, pre-immunised equine peripheral blood mononuclear cells (PBMC) were measured by an indirect enzyme-linked immunoabsorbent assay (ELISA). Optimal anti-TT Ig production occurred at concentrations of stimulating, purified TT of between 0.001 and 0.1 micrograms ml-1, which varied depending on the cell concentration. Optimal anti-TT Ig production was most consistently produced when the cell concentration was 5 x 10(6) ml-1. At this cell concentration maximal anti-TT Ig was induced using 0.1 micrograms ml-1 TT. At a cell c...
Hoshi F, Satho M, Koyama S, Nakadaka K, Chiba M, Ikeda N, Hakamada R, Higuchi S, Kawamura S.The usefulness of a dry-chemistry blood analyzer, Spotchem SP-4410 (SP-4410) in a veterinary clinic for analysis of bovine and equine blood chemistry was studied. We quantitated total protein (TP), albumin (Alb), total bilirubin (T-Bil), blood urea nitrogen (BUN), total cholesterol (T-Cho), glucose (Glu), calcium (Ca), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), creatinine phosphokinase (CPK), and alkaline phosphatase (ALP) in bovine sera. Each sample was assayed with both the SP-4410 and an automated blood analyzer which served as a wet-chemistry reference system, and t...
Lindmark DG, Jarroll EL, Timoney PJ, Shin SJ.The energy metabolism of the English E-CMO strain of contagious equine metritis bacterium was studied in whole cells and cell extracts. This bacterium appears to have an active Krebs cycle and probably obtains energy by oxidative phosphorylation since glycolysis and the hexose monophosphate pathways appear to be absent. These conclusions are based on the findings that [U-14C]glucose incorporation by this bacterium is below the level of detection, and that respiration is stimulated by Krebs cycle intermediates (i.e., malate, citrate, and succinate), but not by glucose, fructose, maltose, or suc...
Schirmer EC, Farooqui J, Polak PE, Szuchet S.Cell-substratum adhesion plays a crucial part in the cascade of events that control growth or turn on and consummate a differentiation program. We are investigating the molecular basis of oligodendrocyte (OLG) cytodifferentiation, employing pure cultures of OLGs isolated from postmyelination brains. We have shown that such OLGs will regenerate in vitro and reenact the ontogenic development of myelin, but to do so they need a signal. Adherence to a polylysine surface in the presence of 20% horse serum generates such a signal. Among the events that are turned on upon OLG adhesion is the phosphor...
Benson CE, Palmer JE, Bannister MF.The antibiograms of 408 Salmonella species isolated from large animals were collected during a three year study from 1981 through 1983. The predominant Salmonella serogroup among these isolates was group B. A consistently high percentage of all isolates were resistant to ampicillin and tetracycline. A pattern of increasing resistance to chloramphenicol and gentamicin was documented for serogroup B isolates while the susceptibility of the isolates to neomycin increased. There was a decrease in the incidence of susceptibility to sulfamethoxazole-trimethoprim among the group E isolates. These cha...
Scare Kenealy JA, Steuer AE.All horses are susceptible to the equine gastrointestinal parasite, Strongylus vulgaris, which is known to cause significant disease and death. The parasite undergoes development from the egg through the first (L1), second (L2) and third (L3) larval stages outside the horse. The L3 is the infective stage. The universally available technique for detection of S. vulgaris larvae is the larval culture method. This requires a 10-14 day culture period to induce development from egg to L3, followed by Baermannization and identification of the L3s to genus and/or species. It is unknown if the culture ...
Millard JT, Chuang E, Lucas JS, Nagy EE, Davis GT.A simple and robust biochemistry laboratory experiment is described that uses restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR) products to verify the identity of a potentially valuable horse. During the first laboratory period, students purify DNA from equine samples and amplify two loci of mitochondrial DNA. During the second laboratory period, students digest PCR products with restriction enzymes and analyze the fragment sizes through agarose gel electrophoresis. An optional step of validating DNA extracts through realtime PCR can expand the experiment to th...
Ripley AM, Penedo MCT, Grahn RA, Martinez de Andino EV, Walbornn SR, Serafini R, Love CC, Hinrichs K.Fetal genotyping has important applications in the horse, but currently necessitates embryo recovery and biopsy. We investigated whether fetal genotyping could be performed on yolk-sac fluid recovered from pregnant mares via transvaginal aspiration. Fluid was collected before Day 30 to provide results before establishment of the endometrial cups (Day 37). Genotyping and assessment of maternal DNA contamination was performed by analyzing histograms of PCR results for 19 loci. In Exp. 1, mares underwent yolk-sac aspiration on Days 22-28 of gestation. Fluid (0.56-1.02 mL) was recovered from fiv...
Arnold C, Chaffin MK, Cohen N, Fajt VR, Taylor RJ, Bernstein LR.To determine the pharmacokinetics of gallium maltolate (GaM) after intragastric administration in adult horses. Methods: 6 adult horses. Methods: Feed was withheld for 12 hours prior to intragastric administration of GaM (20 mg/kg). A single dose of GaM was administered to each horse via a nasogastric tube (time 0). Blood samples were collected at various time points from 0 to 120 hours. Serum was used to determine gallium concentrations by use of inductively coupled plasma-mass spectroscopy. Noncompartmental and compartmental analyses of serum gallium concentrations were performed. Pharmacoki...
Wang ZW, Yu MF, Robinson NE.Muscarinic autoreceptors on horse airway cholinergic nerves were studied by examining the effects of muscarinic receptor antagonists on electrical field stimulation (EFS)-induced acetylcholine (ACh) release in trachealis preparations. All the antagonists including atropine (non-selective), pirenzepine (M1-selective), AF-DX 116 (M2-selective), and hexahydrosiladifenidol (M3-selective) augmented ACh release concentration-dependently. The augmentation was not due to displacement of ACh molecules from tissue receptors into the bath liquid because incubation with atropine after EFS had no influence...
Zibura AE, Cullen MA, Rutledge H, Lassalle L, Salmon JH, Gilger BC, Westermeyer HD.Determine optimal iontophoresis times for riboflavin delivery to the corneal stroma across different species and compare these to corneal injection. Methods: Ex vivo horse, dog, rabbit, and pig globes were treated with riboflavin administered with either iontophoresis for 2.5-20Â minutes with or without corneal epithelium; or with purpose-designed precise corneal injection (PCI) application with intact epithelium. Immediately following riboflavin administration, samples were harvested, frozen, and sectioned. Riboflavin penetration was imaged using fluorescence microscopy. Results: Horse sample...
Smith JM, Meyers KM, Barbee DD, Schott H, Bayly WM.To determine whether plasma von Willebrand factor (vWf) concentration changes in horses during and after treadmill exercise. Methods: 5 mature, fit Thoroughbreds. Methods: A blood sampling catheter was placed in the right jugular vein. A warm-up period was followed by a 3-minute rest period. Horses were galloped at racing pace until fatigued (about 2 minutes). Blood samples were collected prior to warm-up, during the postwarm-up rest period, 1 minute into the run, at cessation of the run, and 5 to 120 minutes after cessation of the run. vWf activity was measured by ELISA and corrected for plas...
Barton MH, Hurley D, Norton N, Heusner G, Costa L, Jones S, Byars D, Watanabe K.Lactoferrin is a colostral glycoprotein with antimicrobial properties. Objective: (1) Serum lactoferrin and immunoglobulin G (IgG) concentrations are correlated and increase in healthy foals after ingestion of colostrum; (2) compared to healthy foals, ill foals will have lower lactoferrin concentrations that correlate with their IgG concentration, neutrophil count, the diagnosis of sepsis, and survival; and (3) plasma concentrations of lactoferrin will be less than serum concentrations. Methods: Healthy foals (n = 16), mature horses (n = 10), and ill foals 1-4 days old (n = 111) that were exam...
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...
Palmisano M, Javsicas L, McNaughten J, Gamsjäger L, Renaud DL, Gomez DE.Anecdotal evidence suggests plasma transfusions increase serum amyloid A (SAA) concentrations in healthy neonatal foals making this marker of inflammation inappropriate for therapeutic decision making in such animals. Objective: Administration of hyperimmune fresh frozen plasma (FFP) increases SAA concentration in healthy foals and in foals with failure of transfer of passive immunity (FTPI). Methods: Eighty-six healthy foals. Methods: Prospective cohort study. Foals <24 hours of age receiving plasma transfusion for treatment of FTPI (serum immunoglobulin G [IgG] concentrations 8 g/L; nÂ...
Read JE, Cabrera-Sharp V, Offord V, Mirczuk SM, Allen SP, Fowkes RC, de Mestre AM.Equine chorionic girdle trophoblast cells play important endocrine and immune functions critical in supporting pregnancy. Very little is known about the genes and pathways that regulate chorionic girdle trophoblast development. Our aim was to identify genes and signalling pathways active in vivo in equine chorionic girdle trophoblast within a critical 7-days window. We exploited the late implantation of the equine conceptus to obtain trophoblast tissue. An Agilent equine 44K microarray was performed using RNA extracted from chorionic girdle and chorion (control) from equine pregnancy days 27, ...
