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.
Creutz C, Sutin N.The kinetics of the reduction of horseheart ferricytochrome c by sodium dithionite (phosphate buffer-sodium chloride; pH 6.5, mu = 1.0, 25 degrees ) features two reaction pathways; one with the rate constant k(3) = 1.17 x 10(4) M(-1) sec(-1), the other with the rate constant k(1)k(2)/k(-1) = 6.0 x 10(4) M(-1) sec(-1). These pathways are interpreted in terms of remote attack (possibly by way of the exposed edge of the porphyrin system) and adjacent attack (requiring the opening of the heme crevice). The limiting rate for the adjacent pathway (k(1) = 30 sec(-1)) is in good agreement with the rat...
Carrier SP, Boulanger P, Bannister GL.The comparative values of the direct, the indirect complement-fixation and the agar-gel immunodiffusion tests were assessed for the diagnosis of equine infectious anemia. Antibodies were detected on the agar-gel immunodiffusion test as early as 18 days post-inoculation in the serums of experimentally infected horses and were readily detectable in all the subsequent bleedings. Complement-fixing antibodies, demonstrable by the direct method, were detected commencing about the same time. However, these were not long-lasting and were replaced by the non-complement-fixing antibodies demonstrable by...
Hart LT, Broussard EA.Immunodiffusion antigen from spleens of horses infected with equine infectious anemia virus was prepared by methods employing freeze-thaw cycles and thiocyanate treatment. Thiocyanate (0.5 M) permitted the recovery of the greatest amount of antigen. Furthermore, it was most effective for recovery of immunodiffusion antigen from spleens which yielded unsatisfactory concentrations of antigen by the conventional freeze-thaw or water-extraction methods. The reactivity of the antigen did not appear to be affected by this chemical treatment.
Larghi OP, González E, Held JR.The corneal test (CT) for rabies diagnosis was evaluated in samples from 313 subjects of different species. Some of the subjects were inoculated experimentally and others were naturally infected. When the CT was compared with immunofluorescence staining and mouse inoculation tests on brains of the same subjects, a sensitivity of 41.7% and a specificity of 100% were found. The authors conclude that a positive CT result would confirm the diagnosis of rabies, but a negative one would not exclude the possibility of disease.
Eguchi M, Kuniyasu C, Kishima M.The passive hemagglutination (PHA) test was improved to enable the detection of antibodies to Taylorella (Haemophilus) equigenitalis in the sera of mares. Horse red blood cells (RBC) fixed with glutaraldehyde were compared with similarly treated RBC of a cow, pig and sheep for the PHA test. The horse RBC were superior to those of the other animals tested in detecting mares affected with contagious equine metritis (CEM). A PHA test using these cells as indicator and an antigen prepared from T. equigenitalis by sonication following treatment with hyaluronidase was the most satisfactory in terms ...
Warlick LM, Léguillette R, Kogan C, Gold JR, Bayly WM.Diagnosis and assessment of severity of exercise-induced pulmonary hemorrhage (EIPH) relies on postexercise visualization of fresh blood in the airways via tracheobronchoscopic examination (TBE) and/or counting erythrocytes in bronchoalveolar lavage fluid (BALFRBC). Determining the BALFRBC is more sensitive than TBE but its usefulness is hampered by the need to have BALFRBC counted at a laboratory. We explored the feasibility of evaluating the severity of EIPH by using a color chart comprised of five shades of red and matching those colors with the color of BALF immediately following collectio...
Brinkmeyer-Langford C, Raudsepp T, Gustafson-Seabury A, Chowdhary BP.A total of 207 BAC clones containing 155 loci were isolated and arranged into a map of linearly ordered overlapping clones over the proximal part of horse chromosome 21 (ECA21), which corresponds to the proximal half of the short arm of human chromosome 19 (HSA19p) and part of HSA5. The clones form two contigs - each corresponding to the respective human chromosomes - that are estimated to be separated by a gap of approximately 200 kb. Of the 155 markers present in the two contigs, 141 (33 genes and 108 STS) were generated and mapped in this study. The BACs provide a 4-5x coverage of the regio...
Vacchina V, Ionescu C, Oguey S, Lobinski R.A method was developed for the quantification of Zn-, Cu- and Mn-glycinates in supplemented feed samples. The coupling of capillary electrophoresis (CE) with ICP MS detection after purification of the extract by ultrafiltration was shown to be efficient for the quantitative recovery of glycinates. The method developed was then applied to evaluate the bioaccessibility of glycinates using a sequential enzymolysis approach. The data obtained indicated a strong bioaccessibility of each element (79-94%). A new complex was also found to be formed during the digestion process. Bioavailability was the...
Pelegrino JL, Vázquez S, Morier L, Castillo A, Guzmán MG, Kourí G.We present the results attained in the identification of Eastern equine encephalomyelitis virus isolations in Vero and XL-2 cell systems, using a double-antibody ELISA technique and the indirect immunofluorescence method. The results attained through these two techniques coincided by 100% with identification through neutralization. With the former, the virus was detected within 6-8 hours after inoculation. Better results were attained with XL-2 cells.
Hunt JP, Haywood PE, Moss MS.A gas chromatographic screening procedure for the non-steroidal anti-inflammatory group of drugs is described. The method invovles on-column methylation of the carboxylic acid group using trimethylanilinium hydroxide as the methylating reagent. Fifteen such drugs were studied. Eight of these were detected in urine collected from horses that had received these compounds orally and for these drugs, rates of urinary excretion are recorded. Seven other members of this group of drugs were shown to be detectable by this procedure but in these cases the drug was added to urine and not administered to...
Singh AK, Mishra U, Ashraf M, Abdennebi EH, Granley K, Dombrovskis D, Hewetson D, Stowe CM.Chemical ionization- and electron impact ionization-selective ion monitoring provided a simple and sensitive method for measuring detomidine (Domosedan), a potent sedative-analgesic drug for horses and cattle. Chemical ionization was at least 10 times more sensitive than electron impact ionization. By using propranolol as an internal standard, we found that the recovery of detomidine from the extraction procedure used in this study was greater than 75% for plasma, whole blood, or urine samples. Approximately 68% of detomidine was bound to plasma protein and 53% was bound to red blood cells.
Lehner AF, Almeida P, Jacobs J, Harkins JD, Karpiesiuk W, Woods WE, Dirikolu L, Bosken JM, Carter WG, Boyles J, Holtz C, Heller T, Nattrass C....Remifentanil (4-methoxycarbonyl-4-[(1-oxopropyl)phenylamino]-1-piperidinepropionic acid methyl ester) is a mu-opioid receptor agonist with considerable abuse potential in racing horses. The identification of its major equine urinary metabolite, 4-methoxycarbonyl-4-[(1-oxopropyl)phenylamino]-1-piperidinepropionic+ ++ acid, an ester hydrolysis product of remifentanil is reported. Administration of remifentanil HCl (5 mg, intravenous) produced clear-cut locomotor responses, establishing the clinical efficacy of this dose. ELISA analysis of postadministration urine samples readily detected fentany...
Apprich V, Spergser J, Rosengarten R, Stanek C.The effects of two dermatophytes (Microsporum gypseum and Trichophyton mentagrophytes) and four moulds (Scopulariopsis brevicaulis, Alternaria alternata, Geotrichum candidum and Penicillium spp.) on living keratinocyte cultures were examined in vitro using primary human keratinocytes. Rates of apoptosis of infected cells were determined using a colorimetric TUNEL system which detects the characteristic nuclear DNA fragmentation of apoptotic cells. The cytotoxicity of the individual fungi was tested by quantitatively measuring cytosolic enzyme lactate dehydrogenase, released upon cell lysis, in...
Schulman ML, Harper CK, Bell E, Nel A, Guthrie AJ.The aim of this study was to establish and validate a method to permit microsatellite analysis of DNA profiles obtained from frozen-thawed stallion sperm cells. This would provide reliable and accurate verification of the identification of a semen donor. Ejaculates from 5 pony stallions were collected, processed and frozen in 0.5 ml plastic straws. Aliquots of 100 microl of the frozen-thawed semen thus obtained were either placed directly, or diluted (1:10; 1:100; and 1:1000) and placed on slides of FTA paper. Similarly, blood samples obtained from each of the stallions were placed onto slides...
Milne EM, Pemberton AD, McGorum BC, Dixon PM, Miller HR.The equine alpha-1 proteinase inhibitor (alpha 1PI) system differs from that of man in that the equine system consists of four closely-linked genes (Spi1-Spi4) whereas in man, a single gene encodes for alpha 1PI. We have previously found differences in the proportion of the Spi proteins in equine serum and bronchoalveolar lavage fluid (BALF). We therefore wished to determine whether, as reported in man, there was any molecular weight difference between the Spi proteins in serum and BALF. alpha 1PI and albumin from equine BALF migrated further towards the anode compared with serum alpha 1PI on ...
Edlund PO, Bowers L, Henion J.Monitoring steroid use requires an understanding of the metabolism in the species in question and development of sensitive methods for screening of the steroid or its metabolites in urine. Qualitative information for confirmation of methandrostenolone and identification of its metabolites was primarily obtained by coupled-column high-performance liquid chromatography-tandem mass spectrometry. The steroids and a sulphuric acid conjugate were isolated and identified by their daughter ion mass spectra in the urine of both man and the horse following administration of methandrostenolone. Spontaneo...
Guan F, Uboh CE, Soma LR, Luo Y, Jahr JS, Driessen B.Oxyglobin (OXY) and Hemopure (HMP) are produced from bovine hemoglobin (Hb) and were developed for the treatment of anemia in animal and human patients, respectively. Hemolink (HML) is a blood substitute of human Hb origin under development. The ability of these agents to carry oxygen in circulating blood and their promise to improve oxygen delivery to tissues supports the potential for their abuse in equine and human athletes. To deter athletes from abuse of these agents, a method has been developed for the detection, confirmation and quantification of OXY, HMP, and HML in equine and human pl...
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...
Marcum JA, Highsmith RF, Kline DL.Complete activation of purified horse plasminogen to plasmin was obtained with a 1:10 molar ratio of streptokinase to plasminogen after 5 min of incubation at 37 degrees C. At a 1:1 molar ratio, maximal activity did not appear until 15-30 min, while at a ratio of 6:1 complete activation was delayed for 120-180 min. Gel filtration studies of isotopically labeled streptokinase and horse plasminogen suggest that the delay was due to impaired formation of a streptokinase-plasminogen complex. The predominant streptokinase moiety within the streptokinase-plasmin complex which forms from the streptok...
Rudolph W, Remane D, Wissenbach DK, Klein C, Barnewitz D, Peters FT.Atypical myopathy (AM) is a fatal disease in horses presumably caused by hypoglycine A (HGA) from ingested maple seeds and its active metabolite methylene cyclopropyl acetic acid (MCPA). The aim of this study was the development and validation of a rapid and simple assay for HGA and MCPA-carnitine in horse serum and its application to authentic samples. Identification and quantification were carried out by ultra high performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS) with full-scan/data-dependent MS/MS. Chromatographic separation was performed by isocrat...
Sun X, Yao H, Zhang C, Lu C.Equine herpesvirus type 1 (EHV-1) is a major cause of respiratory and reproductive diseases in horses worldwide. The genome of EHV-1 strain 438/77 (isolated from an aborted equine fetus) was cloned as a bacterial artificial chromosome (BAC) in E. coli without any gene deletions. The mini-F plasmid sequence was inserted in the middle of ORF19 and 20 via homologous recombination following co-transfection of viral DNA and plasmid pE19_20/HA into RK13 cells. Circular viral DNA was extracted from RK13 cells infected with purified recombinant virus expressing green fluorescent protein (GFP) and elec...
Takehana K, Abe M, Iwasa K, Hiraga T.Complex carbohydrates were examined in glandular cells of the horse duodenal gland by using lectin histochemical techniques. In the horse, the duodenal gland was distributed in the area from the uppermost part of the small intestine to a point about 6m caudal to the pylorus. It consisted of two types of cells, mucous and serous cells. The former was found in glands distributed almost all over this part, but the latter was present in glands distributed restrictedly to the uppermost part of the small intestine at a point about 10 cm caudal to the pylorus. The cytoplasm of the mucous cell contain...
Maher MA, Ahmad IM.The current study was performed on ten adult Egyptian donkeys of both sexes for anatomical and radiographic studies in addition to six other donkeys for enzymatic, biochemical and statistical analysis. The aim was to illustrate the normal hepatic arterial and biliary distribution using different anatomic techniques and radiographic imaging besides, establishing an accurate laboratory profile specific for donkeys that used as standard indicators for hepatobiliary dysfunction. The right branch of the hepatic artery in donkey forms a curved arch erupting five branches, unlike the left branch that...
Craig AM, Blythe LL, Rowe KE, Lassen ED, Walker LL.An oral vitamin E absorption test used in human beings was modified for use in horses. The most appropriate techniques with which to measure gastrointestinal tract absorption of vitamin E (alpha-tocopherol) in horses were developed. Vitamin E was administered orally, and serum values of alpha-tocopherol were measured by use of high-performance liquid chromatography at 0, 3, 6, 9, 12, and 24 hours after vitamin E administration. Variables included comparison of 2 dosages (45 and 90 IU/kg of body weight), routes of administration, and absorption dynamics of 3 preparations of dl-alpha-tocopherol....
Zientara S, Sailleau C.The authors briefly review the clinical forms of equine rhinopneumonitis and indicate changes in the nomenclature of equine herpesviral infections. The value of restriction profiles for epidemiological studies is described, taking as an example the strains of virus isolated in France. A technique is given for preparing molecular probes, as well as the application of these probes in direct diagnosis from biological specimens.
Fuentealba NA, Sguazza GH, Eöry ML, Valera AR, Pecoraro MR, Galosi CM.Equid herpesvirus 1 (EHV-1) infection has a significant economic impact on equine production, causing abortion, respiratory disease, neonatal death and neurological disorders. The identification of specific EHV-1 genes related to virulence and pathogenicity has been the aim of several research groups. The purpose of the present study was to analyze different genomic regions of Argentinean EHV-1 strains and to determine their possible relationship with virulence or clinical signs. Twenty-five EHV-1 Argentinean isolates recovered from different clinical cases between 1979 and 2007 and two refere...
Levina V, Timms M, Vine J, Steel R.A synthetic Interleukin-1 receptor antagonist peptide with the sequence Acetyl-Phe-Glu-Trp-Thr-Pro-Gly-Tyr-Trp-Gln-Pro-Tyr-Ala-Leu-Pro-Leu-OH has been identified in a vial seized during a stable inspection. The use of peptide-based Interleukin-1 receptor antagonists as anti-inflammatory agents has not been previously reported, making this peptide the first in a new class of sports doping peptides. The peptide has been characterized by high-resolution mass spectrometry and a detection method developed based on solid-phase extraction and liquid chromatography - triple quadrupole mass spectrometr...
Mizobe M, Kondo F, Toyoshima C, Kumamoto K, Terada T, Nasu H.A rapid method was developed to analyze delta-bilirubin (B delta), diconjugated bilirubin (DCB), monoconjugated bilirubin (MCB), and unconjugated bilirubin (Bu) by direct injection of sera using high-performance liquid chromatography (HPLC) with an internal-surface reversed-phase silica support (ISRP) column. Sharp bilirubin peaks were obtained using a simple mobile phase of acetonitrile: 0.5 M Tris-HCl buffer (20:80, v/v, pH 7.2). A variable-wavelength detector set at 450 nm, 0.01 absorbance unit full scale (AUFS), and a recorder set at 4 mm/min were used for detection. Peaks for B delta, DCB...
Takeda A, Tanaka H, Shinohara T, Ohtake I.A sample preparation method for mass chromatographic detection of doping drugs from horse plasma is described. Bond Elut Certify (1 g/6 ml) is used for the extraction of 4 ml of horse plasma. Fractionation is performed with 6 ml of CHCl3-Me2CO (8:2) and 5 ml of 1% TEA-MeOH according to its property. Simple and effective clean-up based on non-aqueous partitioning is adopted to remove co-eluted contaminants in both acid and basic fractions. Two kinds of 1-(N,N-diisopropylamino)-n-alkanes are co-injected with the sample into the GC-MS system for the calculation of the retention index. Total recov...
Okumura M, Fujinaga T.The role of keratan sulphate (KS) as a metabolic marker of cartilage was evaluated using an in vitro model of equine articular cartilage. Articular cartilage was harvested from clinically healthy 6-month-old foals (n = 3). Chondrocytes were centrifuged and cultured as pellets. Chondrocyte pellets were stimulated by insulin-like growth factor-I alpha (IGF-I alpha) or interleukin-1 alpha (IL-1 alpha) for 2 weeks. The concentrations of sulphated glycosaminoglycans (GAG) and KS in the culture media were measured by a 1,9-dimethyl-methylene blue (DMMB) colorimetric assay and an inhibition enzyme-li...
Blake JW, Tobin T.Three gas-liquid chromatographic (G.L.C.) procedures discussed have been designed around the four "esses" of detection tests--speed, sensitivity, simplicity, and specificity. These techniques are admirably applicable to the very low plasma drug levels encountered in blood testing under pre-race conditions. The methods are equally applicable to post-race testing procedures, where both blood and urine samples are tested. Drugs can only rarely be detected by the electron capture detector (E.C.D.) without a prior derivatization step, which conveys to the drug(s) high electron affinity. Because of ...
Aberle ED, Judge MD, Kirkham WW, Page EH, Crawford BH.Frozen sections of equine musculus semitendinosus were examined for myosin adenosine triphosphatase (ATPase) and reduced nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR), using standard histochemical procedures, and the proportions of the various fiber types and average fiber sectional size were determined. With ATPase staining, approximately 70% of the fibers were classified as alpha fibers (ATPase positive), and 30%, as beta fibers (ATPase negative). In addition, 2 populations of alpha fibers could be readily distinguished on the basis of the intensity of the ATPase reaction...
Combie J, Blake JW, Nugent TE, Tobin T.We have investigated the action of five sources of beta-glucuronidase enzymes on the hydrolysis of glucuronides of apomorphine, butorphanol, hydromorphone, nalbuphine, oxymorphone and pentazocine in equine urine. For all glucuronides tested, Patella vulgata beta-glucuronidase yielded the largest thin layer chromatographic (TLC) spots. For oxymorphone, P. vulgata was the only treatment to yield detectable TLC spots under test parameters. For these six drugs, TLC spot size and chromatographic quality were compared between control horses and horses pretreated with furosemide four hours earlier. F...
Kingston JK, Bayly WM, Sellon DC, Meyers KM, Wardrop KJ.To investigate the potential use of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-human thrombospondin antibody for detection of the activation of equine platelets by use of flow cytometry. Methods: Platelets obtained from 6 Thoroughbreds. Methods: Flow cytometry was used to assess platelet activation as indicated by detection of binding of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-thrombospondin antibody to unactivated and ADP-, collagen-, platelet activating factor (PAF)-, and A23187-activated equine platelets. Human platelets were used...
