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.
McManus AT, Robinson DM.Reconstituted Venezulean equine encephalitis vaccine was found to retain significant titers of plaque-forming virus after storage at 4 or 22 C for 24 hr.
Momose A, Tsuji T.When sulpyrine and aminopyrine are administered to the horse, unchanged aminopyrine and its metabolites, 4-methylaminoantipyrine and 4-aminoantipyrine, are detected in the urine by means of thin-layer chromatography (TLC) and gas liquid chromatography. Further identification of aminopyrine and these metabolites was carried out by the gas chromatography-mass spectrometry (GC-MS) method. The procedures for separation and identification are as follows : The excretions were adjusted to pH 9 with ammonium hydroxide and extracted with chloroform. The extract was separated by TLC. The spots were loca...
Anderson RS, Day NK, Good RA.Natural hemagglutinin activity against vertebrate erythrocytes is present in the hemolymph of the cockroach Blabarus craniifer. The hemagglutinin titer against rabbit erythrocytes is high, whereas sheep and horse red cells agglutinate weakly. Hemagglutinin activity was depressed by the complement inhibitor, cobra venom factor. Cockroach hemagglutinin is heat-labile; all activity is destroyed by heating at 56 C for 1 hr. A humoral factor similar to the complement component 3 proactivator is also present in cockroach hemolymph. The formation of the cobra venom factor-hemolymph "complex" is depen...
McGuire TC, Van Hoosier GL, Henson JB.The role of non-complement-fixing anti-equine infectious anemia (EIA) antibody in the conversion of complement fixation (CF) tests from positive to negative in EIA-infected horses was investigated. Complement-fixation inhibition (CFI) tests demonstrated antibodies in sera that were CF negative. These antibodies would bind to antigen, but would not fix complement. The inhibiting antibodies were isolated and shown to be IgG(T) by immunoelectrophoresis and immunodiffusion against monospecific anti-IgG(T) antisera. Separation of immunoglobulins from affected horse sera by DEAE cellulose chromatogr...
Herb VM, Url A, Tichy A, Nell B.Establishing an immunohistochemical approach for semi-quantitative assessment of the presence of immunoglobulin G (IgG) in equine, canine, and feline corneas. Methods: Healthy corneas of horses, dogs, and cats, euthanized because of a fatal disease or an unrecoverable trauma unrelated to and without a history of ophthalmic disease were formalin-fixed, paraffin-embedded, and determined to be pathomorphologically healthy by light microscopy. Automated immunohistochemistry was performed using primary antibodies against IgG, biotin-conjugated secondary antibodies, and streptavidin-peroxidase, as w...
Henderson K, Stevens S, Bailey C, Hall G, Stewart J, Wards R.The relative merits of measuring blood concentrations of equine chorionic gonadotrophin (eCG, previously known as pregnant mare serum gonadotrophin (PMSG)), or oestrone sulphate (OS), or faecal OS concentrations for determining pregnancy status in miniature horses were investigated. Pregnant mares between 40 and 140 days after mating had serum eCG concentrations > 1 I.U. mL-1, with the highest concentrations occurring between days 50 and 120. However, eCG measurements were susceptible to returning a 'false positive' diagnosis of pregnancy. Plasma OS concentrations ranged from 0.1 to 3.6 ng ...
Lettry V, Kawasaki H, Sugaya K, Hosoya K, Takagi S, Okumura M.This study aimed to evaluate a system that identifies cartilage turn over and/or degradation through measurement of a new keratan sulfate (KS) epitope concentration in equine sera. Blood samples were collected from 30 horses, 1 (n = 15) and 2 year-olds (n = 15). Serum samples were analyzed for an epitope of keratan sulfate by 1/20/5D4 (KS5D4) and new epitopes of keratan sulfate using high sensitive keratan sulfate (HSKS), measured by two respective enzyme-linked immunosorbant assays (ELISAs). There was no correlation in serum concentration of KS evaluated using 5D4 and HSKS. Age had no signifi...
Bell K, Arthur H, Breen M.Eleven apparent mutations of the equine plasma transferrin and esterase gene (10 in TF and one in ES) were found in an analysis of approximately 240,000 thoroughbred horses. Eight of the transferrin mutations produced variants not previously recognized in horses. In the two remaining transferrin mutations and the esterase mutation, reduced plasma concentrations of the proteins were demonstrated by immunological techniques and together with the family data indicated the existence of 'null' alleles.
Brom-de-Luna JG, Canesin HS, Wright G, Hinrichs K.Nuclear transfer using somatic cells from frozen semen (FzSC) would allow cloning of animals for which no other genetic material is available. Horses are one of the few species for which cloning is commercially feasible; despite this, there is no information available on the culture of equine FzSC. After preliminary trials on equine FzSC, recovered by density-gradient centrifugation, resulted in no growth, we hypothesized that sperm in the culture system negatively affected cell proliferation. Therefore, we evaluated culture of FzSC isolated using fluorescence-assisted cell sorting. In Exp. 1,...
Soma LR, Uboh CE, Guan F, Luo Y, Moate PJ, Boston RC, Driessen B.Hemoglobin-glutamer-200 (HBOC-200) is a hemoglobin (Hb)-based oxygen carrier (HBOC) comprising glutaraldehyde-polymerized bovine Hb. In this study, we sought to determine the pharmacokinetics of this first generation HBOC after IV infusion of 32.5 g of HBOC-200 solution in horses. Quantification of HBOC-200 in equine plasma and urine was performed using a method recently developed by our laboratory. The elimination from plasma was based on size distribution of the bovine Hb polymer. The decline of plasma concentration-time curve of HBOC-200 was described by a noninterchanging 2-compartmental m...
Ortved KF, Goodale MB, Ober C, Maylin GA, Fortier LA.Orthobiologics such as autologous conditioned serum (ACS) are often used to treat joint disease in horses. Because ACS is generated from the horse's own blood, any medication administered at the time of preparation would likely be present in stored ACS, which could lead to an inadvertent positive drug test following intra-articular (IA) injection. The main objective of this study was to determine if ACS prepared from firocoxib positive horses could result in detectable plasma concentrations of the drug following IA injection. Firocoxib was administered to six horses at 0.1mg/kg PO twice at a 2...
Russell KE, Perkins PC, Grindem CB, Walker KM, Sellon DC.To evaluate a method for detecting thiazole orange-positive (TO+, reticulated) platelets in equine blood, using flow cytometry. Methods: 16 healthy, equine infectious anemia virus (EIAV)-negative horses and ponies; 9 thrombocytopenic, EIAV-positive horses and ponies; and 2 thrombocytopenic, EIAV-negative horses. Methods: Blood from healthy and thrombocytopenic horses was collected by jugular venipuncture. Appropriate sample requirement and incubation time for the assay were evaluated, using blood anticoagulated with EDTA or sodium citrate, or platelet-rich plasma in sodium citrate. The sample ...
McKinney AR.Control of the use of performance-affecting substances in the horse is critical to the integrity of a wide range of equine sports, with major implications for both animal welfare and revenue streams. One class of medications enjoying particular public notoriety is the anabolic-androgenic steroid group, as highlighted by the recent 'Big Brown' affair and Congressional inquiries into the use of steroids in professional sports, including horse racing, in the USA. This review examines the latest developments pertaining to the analytical detection of these substances in equine biological samples an...
Goranov Kh.The alkaline phosphatase enzyme, isolated by Morton's method from leukocytes of sheep, goats, and pigs gave after agarose elctrophoresis two isoenzyme fractions moving to the positive pole at the sites of the alpha 1- and alpha 2-globulins of the blood serum. In bovine leukocytes, besides these two fractions there was a third one that moved more slowly in the zone of the beta-globulins. In horses the alkaline phosphatase of leukocytes produced a wide band within the zones of the beta-globulins and the albumins. It was established that the proportion between the individual isoenzyme fractions o...
Roser JF, Evans JW, Mikuckis GM, Adams TE, Hughes JP.As quantified by Scatchard analysis, a 27 000 g crude luteal membrane fraction contained a single population of unoccupied LH receptors characterized by high affinity, ka = 0.647 +/- 0.158 X 10(11) M-1 and low binding capacity, Rt = 4.91 +/- 0.78 X 10(-11) M/mg membrane fraction. Acceptable hormonal specificity, reversibility, saturability, high affinity and tissue specificity indicated that the binding protein was a physiological receptor. To ensure that the methods used for Scatchard analysis were valid, hCG was characterized for specific activity and maximum bindability, non-specific bindin...
Merritt T, Mallonée PG, Merritt AM.Seven healthy foals (five ponies and two horses) were maintained on grass pasture with their dams. All foals had normal faeces at the time of testing. An oral xylose absorption test was performed on each foal at one, two and three months of age. Following an 8 h fast, 0.5 g/kg D-xylose as a 10 per cent solution was given via a nasogastric tube. Control and 30 min interval plasma samples were collected for 3 h and the plasma was analysed for xylose using the phloroglucinol microassay technique. Maximum xylose concentration levels were reached between 30 and 60 mins for each of the foals. The me...
Marshall DE, Mortishire-Smith RJ, Houghton E, Gower DB.Oestradiene-3,17-diol and oestratriene-3,17-diol (or the diol of Heard's ketone (3-hydroxy-5(10),6,8-oestratriene-17-one) have been extracted on a large scale from pooled urines and allantoic fluid obtained from pregnant mares. Initial purification was achieved using column chromatography, and further purification by high performance liquid chromatography or silver nitrate (argentation) thin layer chromatography. The steroids were characterised using gas chromatography-mass spectrometry. Positions of the double bonds in ring B of oestradienediol were deduced on the basis of results of ultravio...
Dhawedkar RG, Jain NC, Mount ME, Bowling AT, Vegad JL.An enzyme-linked immunosorbent assay (ELISA) was standardised and applied for the detection of antiplatelet and antineutrophil antibodies using a heterologous system consisting of equine platelets or neutrophils and antisera raised in rabbits. The standardised technique consisted of using Immulon type 3 plate, 1 per cent gelatine as a blocking solution, poly-L-lysine buffer as a coating solution, unfixed antigen, 90 microliters test serum, horseradish peroxidase conjugated antibody and o-phenylenediamine dihydrochloride as a substrate. The number of unfixed platelets or neutrophils required fo...
Grünig G, Barbis DP, Zhang CH, Davis WC, Lunn DP, Antczak DF.Equine peripheral blood lymphocytes (PBL) were enriched by positive selection using panning with a mixture of monoclonal antibodies against putative equine CD4 (Equine Leucocyte Antigen Workshop antibodies WS 1 and WS 72), or CD8 molecules (Workshop antibodies WS 12, WS 49, and WS 74). RNA was extracted from CD4 enriched cells (99% purity), from CD8 enriched cells (69% purity), from peripheral blood lymphocytes, and from neonatal equine thymus. RNA extracted from equine granulocytes and from equine kidney served as negative control. The RNA was electrophoresed in agarose and transferred to nyl...
Wu H, Bi X, Cao F, Zhu C, Liu H, Song J, Ma L, Ma L, Zhang Y, Zhao D, Liu H, Xu X, Zhang S.A proliferation inducing ligand (APRIL) is a member of the TNF superfamily. It shares two receptors with B-cell activating factor (BAFF), B-cell maturation antigen (BCMA), and transmembrane activator and CAML interactor (TACI). Herein, the equine APRIL was identified from equine adipose-derived stem cell (ASC), and the protein expression of APRIL and its related molecules were detected during the adipogenic differentiation of equine ASC in vitro. The equine APRIL gene was located on chromosome 11, spans 1852 base pairs (bp). Its open reading frame covers 753 bp, encoding a 250-amino acid prot...
Ganjam VK, Kenney RM, Flickinger G.The pattern of plasma progestagen levels during the oestrous cycle was similar to that previously reported except for lower peak levels. The lack of significant difference (p less than 0-01) between CPBA and RIA values suggests that progesterone itself is the major component during the oestrous cycle. Progesterone levels during the first and second post-parturient oestrous cycles were similar to those observed during the cycle of the non-pregnant mare. During pregnancy there were two peaks of plasma progestagens. The first, which occurred during the 3rd month, coincided with high levels of PMS...
Barnes RJ, Comline RS, Jeffcott LB, Mitchell MD, Rossdale PD, Silver M.The concentrations of 13, 14-dihydro-15-oxo-prostaglandin F(PGFM), the stable metabolite of prostaglandin F, were measured in the plasma of catheterized mares and foetuses and non-catheterized thoroughbred mares and ponies during the last months of gestation. The plasma concentration of PGFM increased gradually towards term in all groups of animals. During the operation for insertion of catheters, maternal and foetal concentrations of PGFM were high, but the values fell to basal levels 24--48 h after the operation. It was found the preoperative starvation (24 h) led to a rise in the concentrat...
Cambridge H, Reynoldson JA, Dunsmore JD, Hilbert BJ.A radioimmunoassay for thromboxane B2 (TXB2) in unextracted horse plasma was evaluated. Sensitivity of the assay was 14.0 (SD 5.6) pg ml-1 of plasma. Interassay and intra-assay variation were 21.3 per cent and 4.3 per cent, respectively. The percentage of tracer bound in unextracted plasma in the absence of TXB2 was often higher than that in buffer. Therefore standard curves were obtained using standards diluted in plasma from horses treated with aspirin or in charcoal treated TXB2-free plasma. Standard curves determined in plasma and buffer were parallel. This assay was used to determine the ...
Schlichtherle C.This paper describes the importance of fumonisins for human beings and animals and shows data for the occurence in food. Corn-based food samples (n = 299) purchased in the area of munich were analyzed for fumonisin content using an enzyme immunoassay.Fumonisins are mycotoxins produced byFusarium species, especially byFusarium moniliforme andFusarium proliferatum. Occurrence of fumonisins in corn and in cornbased foods and feeds has been reported from almost all over the world. In several animal species different diseases are traced back to fumonisin toxicosis. Fumonisin levels of 5-10 ppm inho...
Nouws JF.As part of the examination of emergency-slaughtered animals for the presence of antibiotic residues, studies were done to see whether false-positive results would be obtained when the Sarcina lutea kidney test and Bacillus subtilis BGA test were performed. When the S. lutea kidney test was positive in cattle, calves and swine, penicillin was invariably found to be present in those animals, the histories of which showed that they had not been given antibiotics. A syringe and an injected fluid containing penicillin residues are regarded as possible causes of these positive results. When the S. l...
Stanley SM, Wilhelmi BS, Rodgers JP, Bertschinger H.Immunoaffinity chromatography using a synthesised immunosorbent was used to extract tritiated dexamethasone (with dexamethasone carrier) from equine urine at a recovery of 81.7 +/- 8.4% (mean +/- S.D.). A method utilising this procedure coupled to cool on-column injection gas chromatography-negative ion chemical ionisation mass spectrometry is also described for the confirmation of low levels of flumethasone in equine urine samples.
Pellegrini A, Hägeli G, von Fellenberg R.1. Trypsin digestion of perchloric acid precipitated horse plasma yielded polypeptides with inhibitory properties for trypsin, chymotrypsin and, to a small extent, kallikrein. 2. The Mr of the inhibitory polypeptides were 73,000 and 24,000. 3. The number, enzyme specificity and Mr of the inhibitory polypeptides differed from the values known for the human being. 4. The inhibitory polypeptides were purified by affinity chromatography on Sepharose-trypsin and by gel filtration through Sephadex G-75. 5. Protease inhibitory polypeptides were generated in the same manner by chymotrypsin, elastase, ...
Bailey E, Lear TL.We compared pools of DNA from 10 Thoroughbred horses and 10 Arabian horses for the presence of randomly amplified polymorphic DNA (RAPD) markers which might be useful in distinguishing between the breeds. Using 212 decamer oligonucleotides and our polymerase chain reaction (PCR) conditions, 173 of the primers produced scoreable bands. The number of bands ranged from 0 to 9 with an average of 3.6. In family studies using 11 arbitrarily selected primers, five of the 11 primers produced polymorphic bands which exhibited Mendelian inheritance as dominant markers. When comparing the pooled DNA from...
Schultheiss PC, Collins JK, Carman J.An indirect immunoperoxidase (IP) procedure using the avidin-biotin-peroxidase complex detection technique was developed to detect viral equine herpesvirus-1 (EHV-1) antigen in formalin-fixed paraffin-embedded tissues from aborted equine fetuses. The procedure was applied to liver, lung, and other tissues from 20 cases of confirmed or suspected EHV-1-induced abortions. Specific staining was observed in tissue sections from EHV-1-infected fetuses. Positive IP staining was present in tissues of 7 cases that were also positive by fluorescent antibody (FA) and virus isolation (VI) and that had typ...
Appolonova SA, Baranov PA, Mesonzhnik NV, Brazhnikova DO, Rodchenkov GM.A method is described for the determination of mesocarb abuse in equestrian sport by combining gradient liquid chromatography and electrospray ionization tandem mass spectrometry. Mesocarb was administrated orally to two horses at a dose of 50 µg/kg. Urine samples were collected up to 120 h post administration. Hydrolyzed and conjugated urine fractions were handled using liquid-liquid extraction (LLE). The identity of the parent drug and metabolites was confirmed using liquid chromatography combined with tandem mass spectrometry (MS/MS). Mesocarb and seven metabolites were detected in horse...
Rosskopf U, Noeske K, Werner E.The bacterium Clostridium (C.) tetani is an ubiquitous pathogen. This anaerobic, gram-positive bacterium can form spores and can be found in the whole environment. It enters the body via injuries of the skin and wounds where it releases the neurotoxin "tetanospasmin" (= tetanus toxin). The animals most susceptible to tetanus infection are horses and sheep. Only active immunisation by tetanus vaccine provides effective protection against tetanus intoxication. The marketing authorisation requirements stipulate that efficacy of tetanus vaccines ad us. vet. must be demonstrated in all target anima...
Brown CM, Sonea I, Nachreiner RF, Obradovich JE.Using commercially available diagnostic reagents, serum immunoreactive gastrin activity was measured in five normal horses that were starved of food and water for 24 hours. Blood samples were taken every 15 minutes for two hours. The horses were then fed a pelleted diet for 15 minutes and samples were taken every 15 minutes for a further two hours. Three further samples were taken at hourly intervals. The total sampling period was seven hours. Basal immunoreactive gastrin activity was lower than that reported in other mammals, ranging from a mean of 7.0 pg/ml to 13.8 pg/ml. At 30, 60 and 75 mi...
Poyato-Bonilla J, Anaya-Calvo G, Molina A, Valera M, Moreno-Millán M, Dorado J, Demyda-Peyrás S.Chromosomal abnormalities are a major cause of infertility and reproductive problems in equids. Nowadays, their detection is rising due to the use of new diagnostic tools based on molecular markers instead of karyotyping. Reports of this kind of genetic aberrations in domestic donkeys (Equus asinus) are extremely scarce, despite their importance in human activities. In the present study, we analysed the implementation of a short-tandem-repeat (STR)-based molecular method initially developed for horses, as a diagnostic tool to detect chromosomal abnormalities in donkeys. The frequency of five X...
Sojka JE, Levy M.This article outlines strategies on how to approach equine endocrine disorders based on clinical signs and clinical pathologic data. In the 1987 Veterinary Clinics of North America: Equine Practice article on evaluating equine endocrine function, Beech stated that the numbers of hormonal assays available to use in horses was limited. Unfortunately, not much has changed since then. With the advent of convenient assay kits for many hormones and cofactors available in human medicine, it is possible to submit samples to laboratories for measurement of a wide range of endogenous substances. Caution...
