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.
Mansfield LS, Schott HC, Murphy AJ, Rossano MG, Tanhauser SM, Patterson JS, Nelson K, Ewart SL, Marteniuk JV, Bowman DD, Kaneene JB.Sarcocystis neurona is a protozoan parasite that can cause neurological deficits in infected horses. The route of transmission is by fecal-oral transfer of sporocysts from opossums. However, the species identity and the lifecycle are not completely known. In this study, Sarcocystis merozoites from eight isolates obtained from Michigan horses were compared to S. neurona from a California horse (UCD1), Sarcocystis from a grackle (Cornell), and five Sarcocystis isolates from feral opossums from Michigan. Comparisons were made using several techniques. SDS-PAGE analysis with silver staining showed...
Marsh AE, Mullins AL, Lakritz J.Parasite-specific incorporation of (3)H-uracil was used to assess the replication of Sarcocystis neurona, a protozoal parasite associated with equine protozoal myeloencephalitis (EPM). Anti-protozoal drugs, pyrimethamine (0.01, 0.1 and 1.0microg/ml PYR), sulfadiazine (5microg/ml; SDZ), sulfamethoxazole (5microg/ml; SMZ), diclazuril (100ng/ml; DCZ), atovaquone (0.04ng/ml; ATQ), tetracycline (5microg/ml; TET) and the herbicide glyphosate (1.5 and 4.5mM; GLY) were studied with varying S. neurona parasite densities (2x10(1)-1.2x10(6)merozoites/well). A microtiter plate format was used to test thes...
The Journal of parasitologyFebruary 24, 2001
Volume 86, Issue 6 1366-1368 doi: 10.1645/0022-3395(2000)086[1366:ARAPDP]2.0.CO;2
Spencer JA, Witherow AK, Blagburn BL.Neospora caninum is a recently described coccidial parasite that was first isolated from a dog in 1988 and has subsequently been shown to infect a wide range of mammals. Neospora hughesi, a new species of this genus, has recently been isolated from the spinal cord of horses showing clinical signs of equine protozoal myeloencephalitis. The random amplified polymorphic DNA polymerase chain reaction technique is capable of differentiating between N. caninum and N. hughesi.
Magnarelli LA, Ijdo JW, Van Andel AE, Wu C, Fikrig E.To develop and evaluate a polyvalent ELISA incorporating a highly specific recombinant antigen (p44) for diagnosis of granulocytic ehrlichiosis in dogs and horses. Methods: 32 dogs and 43 horses. Methods: Results of the ELISA were compared with results of indirect fluorescent antibody (IFA) staining and western immunoblotting incorporating whole-cell antigen. Results: For the canine and equine samples, percentages of samples with positive IFA staining, western immunoblotting, and ELISA results were similar. For 29 (91 %) canine samples and 30 (70%) equine samples, results of IFA staining, west...
Hooper PT, Williamson MM.The most important clinical and pathological manifestation of Hendra virus infection in horses and humans is that of severe interstitial pneumonia caused by viral infection of small blood vessels. The virus is also capable of causing nervous disease. Hendra virus is not contagious in horses and is spread by close contact with body fluids, such as froth from infected lungs. Diagnosis should be based on the laboratory examination of blood, lung, kidney, spleen, and, if nervous signs are present, also of the brain. Evidence of infection with the more recently identified and related Nipah virus wa...
Williams SM, Fulton RM, Patterson JS, Reed WM.The diagnosis of eastern equine encephalitis (EEE) virus infection in avian species is relatively difficult when compared with other species. There are no characteristic histologic lesions in the avian brain that would serve to distinguish EEE from infections with, for example, Newcastle disease or highly pathogenic avian influenza virus. Traditionally, virus isolation (VI) and/or hemagglutination inhibition (HI) has been used for a definitive diagnosis of EEE in birds. Recently, we developed an immunohistochemistry (IHC) technique for confirmatory diagnosis of EEE infection in equine brain. T...
Baskurt OK, Bor-Kucukatay M, Yalcin O, Meiselman HJ.Differences of red blood cell (RBC) aggregation among various mammalian species has been previously reported for whole blood, for RBC in autologous plasma, and for washed RBC re-suspended in polymer solutions. The latter observation implies the role of cellular factors, yet comparative studies of such factors are relatively limited. The present study thus investigated RBC aggregation and RBC electrophoretic mobility (EPM) for guinea pigs, rabbits, rats, humans and horses; RBC were re-suspended in isotonic 500 kDa dextran solutions for the EPM and aggregation measurements, with aggregation stud...
Donahue JM, Williams NM.The clinical signs, laboratory findings, diagnosis, epidemiology, treatment, and prevention and control of two emerging causes of placentitis and abortion in horses are described in this article. Leptospirosis has been reported as a significant cause of fetal loss in horses in Kentucky, Northern Ireland, and England. Most abortions result from infection by serovars kennewicki or bratislava. Nocardioform placentitis has become the most common cause of placentitis in central Kentucky horses. Nocardioform placentitis is associated with infection by unnamed, gram-positive, filamentous, branching b...
Merkies K, Chenier T, Plante C, Buhr MM.Viability of spermatozoa can be assessed by numerous methods, but many are slow and poorly repeatable, and subjectively assess only 100 to 200 spermatozoa per ejaculate. We collected two ejaculates from each of 4 stallions, and extended them to 50x10(6) sperm/mL in a nonfat dried milk solids glucose extender (EZ Mixin). Half the ejaculate was freeze-killed by immersing in liquid nitrogen for 10 min. Aliquots using appropriate volumes of live and freeze-killed spermatozoa provided the following ratios of live:dead spermatozoa: 100:0, 75:25, 50:50, 25:75, 0:100. We determined the viability of ea...
Raudsepp T, Christensen K, Chowdhar BP.With the expansion of comparative genome analysis across different mammals, there is an increasing need to have well-defined banded karyotypes for the species chosen for investigation. In this context, the steadily growing gene mapping data in the donkey urgently require a framework whereby alignment/comparison of genetic information can be readily made with equids and other mammalian species. Hence a GTG-banded karyotype of the donkey (Equus asinus; EAS) is presented, along with schematic drawings and nomenclature of the banded chromosomes. In addition, the most characteristic features of ind...
Stewart JM, Blakely JA, Johnson MD.Non-covalent modification of cytochrome c may have implications for electron transport and energy metabolism. We examined the interaction of various fatty acids (FAs), their coenzyme A and carnitine esters, and fatty alcohols with horse heart ferrocytochrome c. A comparison of FAs indicated a minimum chain length of 14 carbons was required for significant effect on the ferroheme chromophore and major changes in electronic spectra. Coenzyme A and carnitine esters interacted less strongly than FAs whereas long-chain alcohols did not interact with the protein. We found a single, saturable FA bind...
Powell DG.Examples of equine disease surveillance at the local, regional, national and international level are discussed in this article. Reporting systems at each level are also considered, and the increasing importance of laboratory confirmed diagnoses is emphasized. The need to develop national disease reporting systems is addressed, particularly with respect to conforming to international trading standards.
Mead DG, Ramberg FB, Maré CJ.In previous experiments we have demonstrated that colonized and wild black flies are competent laboratory vectors of different Mexican and Western USA isolates of vesicular stomatitis virus, serotype New Jersey (VSV-NJ). We have recently demonstrated biological VSV-NJ transmission by black flies using animal models. In the study described here, we tested the vector competence of colonized and wild black flies for the vesicular stomatitis virus, serotype Indiana (VSV-IN). A 1998 equine isolate was used. After a 10 day incubation period, saliva from experimentally infected Simulium vittatum and ...
The Journal of parasitologyFebruary 24, 2001
Volume 86, Issue 6 1276-1280 doi: 10.1645/0022-3395(2000)086[1276:COTLCO]2.0.CO;2
Dubey JP, Saville WJ, Lindsay DS, Stich RW, Stanek JF, Speert CA, Rosenthal BM, Njoku CJ, Kwok OC, Shen SK, Reed SM.Sarcocystis neurona is the most important cause of a neurologic disease in horses, equine protozoal myeloencephalitis (EPM). The complete life cycle of S. neurona, including the description of sarcocysts and intermediate hosts, has not been completed until now. Opossums (Didelphis spp.) are definitive hosts, and horses and other mammals are aberrant hosts. In the present study, laboratory-raised domestic cats (Felis domesticus) were fed sporocysts from the intestine of a naturally infected opossum (Didelphis virginiana). Microscopic sarcocysts, with a maximum size of 700 x 50 microm, developed...
Venugopal CS, Holmes EP, Koch CE, Curtis LA, Holm AS, Moore RM.To evaluate the effectiveness of 2 potential endothelin (ET)-1 antagonists in blocking the contractile responses of equine colonic vessels to increasing concentrations of ET-1. Methods: Mesenteric vessels from 6 clinically healthy horses. Methods: Colonic vessels (arterial and venous rings) were placed in organ baths with oxygenated Tyrode solution at 37 C. Each was attached to a force transducer interfaced with a polygraph, and 2 g of tension was applied and equilibrated for 45 minutes. Then, B-1 (PD 142893) and B-2 (PD 145065) ET-1 antagonists were tested. One ring from each vessel type was ...
Hagen SJ, Carswell CW, Sjolander EM.We have measured the effect of temperature and denaturant concentration on the rate of intrachain diffusion in an unfolded protein. After photodissociating a ligand from the heme iron of unfolded horse cytochrome c, we use transient optical absorption spectroscopy to measure the time scale of the diffusive motions that bring the heme, located at His18, into contact with its native ligand, Met80. Measuring the rate at which this 62 residue intrachain loop forms under both folding and unfolding conditions, we find a significant effect of denaturant on the chain dynamics. The diffusion of the cha...
Tobaly-Tapiero J, Bittoun P, Neves M, Guillemin MC, Lecellier CH, Puvion-Dutilleul F, Gicquel B, Zientara S, Giron ML, de Thé H, Saïb A.Foamy viruses (FVs) are complex retroviruses which have been isolated from different animal species including nonhuman primates, cattle, and cats. Here, we report the isolation and characterization of a new FV isolated from blood samples of horses. Similar to other FVs, the equine foamy virus (EFV) exhibits a highly characteristic ultrastructure and induces syncytium formation and subsequent cell lysis on a large number of cell lines. Molecular cloning of EFV reveals that the general organization is that of other known FVs, whereas sequence similarity with its bovine FV counterpart is only 40%...
Kim SK, Buczynski KA, Caughman GB, O'Callaghan DJ.The equine herpesvirus 1 (EHV-1) immediate-early (IE) phosphoprotein is essential for the activation of transcription from viral early and late promoters and regulates transcription from its own promoter. The IE protein of 1487 amino acids contains a serine-rich tract (SRT) between residues 181 and 220. Deletion of the SRT decreased transactivation activity of the IE protein. Previous results from investigation of the ICP4 protein, the IE homolog of herpes simplex virus 1 (HSV-1), revealed that a domain containing a serine-rich tract interacts with EAP (Epstein-Barr virus-encoded small nuclear...
Dohmann K, Wagner B, Horohov DW, Leibold W.In the present study, we describe the expression of equine IL2 and IL4. The cDNA of equine IL2 or IL4 was cloned in a mammalian expression vector, containing c-terminal myc- and six histidines His(6)-epitopes for recognition and purification of equine cytokines. The vector constructs were used for transfection of chinese hamster ovary (CHO) cells. Purified equine cytokines were characterised by western blotting. Equine IL2 was secreted with a molecular weight of approximately 17.1kDa, whereas IL4 was expressed in three different sizes of 17.1, 19.6 and 22.1kDa, probably due to different glycos...
Bousfield GR, Butnev VY, Butnev VY.The O-glycosylation sites for equine LHss (eLHss) and eCGss were identified by solid-phase Edman degradation of four glycopeptides derived from the C-terminal region. Both subunits were O-glycosylated at the same 12 positions, rather than the 4-6 sites anticipated. These sites were partially glycosylated, with carbohydrate attachment ranging from 20% to 100% for eCGss and from 10% to 100% for eLHss. When the C-terminal peptide containing all but one of the O-linked oligosaccharides was removed by mild acid hydrolysis of either eLHss or eCGss, hybrid hormones could be obtained by reassociating ...
Pietra M, Guglielmini C, Forni M, Cinotti S.The viscosity of the mucus, its DNA concentration and the size range of the DNA were determined on tracheobronchial samples from 11 horses with lower airway diseases before and after incubation with recombinant human deoxyribonuclease (rhDNase). The horses were divided into two groups on the basis of the cytology of the samples: group A (five horses) with more than 60 per cent neutrophils and group B (six horses) with fewer than 50 per cent neutrophils. The mean mucus viscosity and DNA concentration in the preincubation samples were significantly higher in group A than in group B, and there wa...
Godard S, Vaiman A, Schibler L, Mariat D, Vaiman D, Cribiu EP, Guérin G.The purpose of this study was to increase the number of genes assigned by in situ hybridization to equine chromosomes and thus the number of links for comparative mapping with other species. Forty-four new sequences were added to the horse cytogenetic map by FISH mapping of BAC clones containing genes (35) or ESTs (9). Three approaches were developed: use of horse BAC clones screened with (i) horse EST primers, (ii) interspecific consensus intraexonic primers, and (iii) use of goat BAC containing genes previously localized on goat chromosomes. Present data suggest that the second approach is t...
Bulone V, Rademaker GJ, Pergantis S, Krogstad-Johnsen T, Smestad-Paulsen B, Thomas-Oates J.Separation of horse dander allergens using two-dimensional PAGE resulted in the identification of 16 proteins that react with allergic patient sera. A sensitive method has been developed for analysing the structures of the glycan chains of individual glycoprotein allergens transferred to blots following two-dimensional PAGE, and has allowed the structural identification of the glycan chains of the most abundant isoforms of Equ c 1, a glycosylated horse dander major allergen. The method involves separation of the allergens by two-dimensional PAGE, transfer to polyvinylidene difluoride membranes...
Wasfi IA, Abdel Hadi AA, Elghazali M, Boni NS, Alkatheeri NA, Barezaig IM, Al Muharami AM, Hamid AM.The pharmacokinetics of tripelennamine (T) was compared in horses (n = 6) and camels (n = 5) following intravenous (i.v.) administration of a dose of 0.5 mg/kg body weight. Furthermore, the metabolism and urinary detection time was studied in camels. The data obtained (median and range in brackets) in camels and horses, respectively, were as follows: the terminal elimination half-lives were 2.39 (1.91-6.54) and 2.08 (1.31-5.65) h, total body clearances were 0.97 (0.82-1.42) and 0.84 (0.64-1.17)L/h/kg. The volumes of distribution at steady state were 2.87 (1.59-6.67) and 1.69 (1.18-3.50) L/kg, ...
Murphy DJ, Todhunter RJ, Fubini SL, Vernier-Singer M, Straubinger RK, Lust G.To study in vitro (1) the dose-response relationships between proteoglycan metabolism in normal and corticosteroid-treated articular cartilage; (2) long-term proteoglycan metabolism after treatment of articular cartilage with corticosteroids; and (3) the effect of corticosteroids on proteoglycan metabolism in articular cartilage treated with monocyte-conditioned medium (MCM). Methods: Equine and canine articular cartilage explants were treated with corticosteroids and MCM. Proteoglycan synthesis and degradation were measured by radioactive labeling in short-term culture, and the long-term effe...
Hinton TM, Li F, Crabb BS.Equine rhinitis A virus (ERAV) has recently been classified as an aphthovirus, a genus otherwise comprised of the different serotypes of Foot-and-mouth disease virus (FMDV). FMDV initiates translation via a type II internal ribosomal entry site (IRES) and utilizes two in-frame AUG codons to produce the leader proteinases Lab and Lb. Here we show that the ERAV 5' nontranslated region also possesses the core structures of a type II IRES. The functional activity of this region was characterized by transfection of bicistronic plasmids into BHK-21 cells. In this system the core type II structures, ...
Guan F, Uboh CE, Soma LR, Birks EK, Teleis D, Rudy JA, Watson AO, Tsang DS.A reliable and sensitive method for the extraction and quantification of phenytoin (5,5'-diphenylhydantoin), its major metabolite, 5-(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) and minor metabolite, 5-(m-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) in horse urine and plasma is described. The method involves the use of solid-phase extraction (SPE), liquid-liquid extraction (LLE), enzyme hydrolysis (EH) and high-performance liquid chromatography (HPLC). The minor metabolite, 5-(m-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) was not present in a reliably quantifiable concentration in all samples. The ...
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...
Stockert JC, Trigoso CI, Tato A, Ferrer JM.The structure of specific granules from horse eosinophil leukocytes is still largely unknown. In this work, electron microscopical studies of horse eosinophils reveal that the large cytoplasmic granules contain an external membrane, a matrix of less density, and a dense (non crystalline) core. Round vacuolar inclusions of matrix materials were often observed within the cores. Horse eosinophil granules showed a considerable heterogeneity, and three morphological types could be identified according to structural features of the core and matrix.
Popot MA, Ho ENM, Stojiljkovic N, Bagilet F, Remy P, Maciejewski P, Loup B, Chan GHM, Hargrave S, Arthur RM, Russo C, White J, Hincks P, Pearce C....Cobalt is an essential mineral micronutrient and is regularly present in equine nutritional and feed supplements. Therefore, cobalt is naturally present at low concentrations in biological samples. The administration of cobalt chloride is considered to be blood doping and is thus prohibited. To control the misuse of cobalt, it was mandatory to establish an international threshold for cobalt in plasma and/or in urine. To achieve this goal, an international collaboration, consisting of an interlaboratory comparison between 5 laboratories for the urine study and 8 laboratories for the plasma stud...
Anestad G, Maagaard O.During an epizootic of equine influenza in Norway caused by influenza A/equine (H3N8) virus the efficacy of rapid virus diagnosis by the indirect immunofluorescence technique was evaluated. The antiserum used in the test was a polyclonal influenza A virus antiserum with reactivity directed mainly against the common nucleoprotein and matrix protein. This antiserum possessed sufficient reactivity for the detection of virus-infected exfoliated nasopharyngeal cells. Nasopharyngeal smear samples from 92 horses were examined and a positive diagnosis was obtained for 57 (62 per cent). Paired serum sa...
Yu NH, Ho EN, Tang FP, Wan TS, Wong AS.A multi-target high-throughput liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the detection of low ppt to low ppb levels of anabolic steroids, corticosteroids, anti-diabetics, and non-steroidal anti-inflammatory drugs (NSAIDs) in equine plasma was developed for the purpose of doping control. Plasma samples were first deproteinated by addition of trichloroacetic acid. Drugs were then extracted by solid-phase extraction (SPE) using Bond Elut Certify cartridges, and the extracts were analysed by a triple-quadrupole/linear ion trap LC-MS-MS instrument in positive electrospray...
Scott TW, Olson JG, All BP, Gibbs EP.Sensitivity and specificity of an antigen-capture ELISA vs virus isolation in cell culture were evaluated for the detection of eastern equine encephalomyelitis (EEE) virus in the brain tissue of naturally infected equids. Brain specimens from 16 equids with neurologic disease were examined by ELISA and by inoculation onto baby hamster kidney cell cultures. Of 10 brain samples from which virus was isolated in the cell culture bioassay, all were correctly identified as containing EEE virus antigen by ELISA. None of the remaining 6 specimens, without detectable infectious EEE virus, contained det...
Machtinger ET, Geden CJ.House flies (Musca domestica L.) and stable flies (Stomoxys calcitrans (L.)) (Diptera: Muscidae) are common pests in equine and cattle facilities. Pupal parasitoids, primarily in the genera Spalangia and Muscidifurax (Hymenoptera: Pteromalidae), can be purchased for biological control of these flies. However, little is known about the host-habitat preferences associated with host-seeking by these parasitoids. The preferences of two Spalangia and two Muscidifurax species to odors associated with house fly hosts in equine and bovine manure were investigated in the laboratory using a Y-tube olfac...
Caetano AR, Lyons LA, Laughlin TF, O'Brien SJ, Murray JD, Bowling AT.Comparative anchor tagged sequences (CATS) from human Chromosome 5 (HSA5) were used as PCR primers to produce molecular markers for synteny mapping in the horse. Primer sets for 21 genes yielded eight horse-specific markers, which were mapped with the UC Davis horse-mouse somatic cell hybrid panel into two synteny groups: UCD14 and UCD21. These data, in conjunction with earlier human chromosome painting studies of the horse karyotype and synteny mapping of horse microsatellite markers physically mapped by FISH, confirm the assignment of UCD21 to ECA21 and suggest that UCD14 is located on ECA14...
Ceusters JD, Serteyn DA, Minguet G, de la Rebière de Pouyade G, Romainville J, Deby-Dupont GP, Mouithys-Mickalad AA, Franck TJ.Horses are particularly sensitive and exposed to excessive inflammatory responses evolving toward an important stimulation of polymorphonuclear neutrophils (PMNs). The aim of this work was to stimulate equine neutrophils in whole blood and to evaluate their response by measuring the release of total and active myeloperoxidase (MPO) and total elastase, considered as markers of neutrophil stimulation and degranulation. Because of the critical importance of the concomitant presence of LPS and TNF-α in equine pathological situations, we combined these two natural mediators to stimulate PMN and co...
Ortiz I, Felix M, Resende H, Ramírez-Agámez L, Love CC, Hinrichs K.To define the effect of sperm agglutination, associated with incubation under capacitating conditions, on accuracy of membrane assessment via flow cytometry and to develop methods to mitigate that effect. Methods: Sperm motility was measured by CASA. Sperm were stained with PI-PSA or a novel method, LD-PSA, using fixable live/dead stain and cell dissociation treatment, before flow-cytometric analysis. Using LD-PSA, acrosome reaction and plasma membrane status were determined in equine sperm treated with 10 μm A23187 for 10 min, followed by 0, 1, or 2 h incubation in capacitating conditions. R...
Goudet G, Belin F, Młodawska W, Bézard J.The effect of epidermal growth factor (EGF) on the in vitro maturation rate of equine oocytes was examined. Oocytes were collected from an abattoir (Expt 1) or using ultrasound-guided follicular puncture in vivo (Expt 2). All oocytes with a compact or expanded cumulus at recovery were cultured for 30 h in: medium 1 (TCM199 + fetal calf serum (FCS) + crude equine gonadotrophin (CEG) + oestradiol + antibiotics); medium 2 (TCM199 + EGF); medium 3 (medium 1 without FCS + EGF); or medium 4 (medium 1 without CEG + EGF). In Expt 1, 84% (37/44) and 87% (40/46) cumulus expansion (P > 0.05), and 39% (22...
Zhang K, Zhou R, Huang H, Ma W, Qi Y, Li B, Zhang D, Li K, Chu H.The aim of this study was to identify the aggregation sites and transmission characteristics of Gasterophilus pecorum, the dominant pathogen of endangered equines in desert steppe. Therefore, we tested with a four-arm olfactometer the olfactory response of the G. pecorum adults to the odors that have a great impact on their life cycle, and also investigated the occurrence sites of the adults in the area where the Przewalski's horse (Equus przewalskii) roam frequently during the peak period of G. pecorum infection. The results of four-directional olfactory test showed that the fresh horse feces...
Aguilera-Tejéro E, Pascoe JR, Amis TC, Kurpershoek CJ, Woliner MJ.A rebreathing method for measurement of pulmonary diffusing capacity for carbon monoxide (DLCO) and functional residual capacity (FRC) was evaluated in conscious horses. Horses were manually ventilated through an endotracheal tube, using a custom-made syringe filled with a gas mixture containing 18-carbon monoxide (18CO) and helium (He). The 18CO and He concentrations were continuously monitored by use of a mass spectrometer connected to the rebreathing circuit. Values for DLCO and FRC were calculated from changes in the concentration of these 2 gases. In 11 Thoroughbreds, mean (+/- SD) DLCO w...
Yu NH, Ho EN, Leung DK, Wan TS.Anabolic steroids have the capability of improving athletic performance and are banned substances in the Olympic games as well as in horseracing and equestrian competitions. The control of their abuse in racehorses is traditionally performed by detecting the presence of anabolic steroids and/or their metabolite(s) in urine samples using gas chromatography-mass spectrometry (GC-MS). However, this approach usually requires tedious sample processing and chemical derivatisation steps and could be very insensitive in detecting certain steroids. This paper describes a high performance liquid chromat...
Thorsteinsdóttir L, Torsteinsdóttir S, Svansson V.Due to the slow growth of equine gammaherpesviruses, isolation of these viruses requires cells that can be propagated long term and show clear cytopathy following infection. Equine cell lines with extended lifespan were established from primary cells originating from equine fetal kidney and lung by transfecting the cells with the retroviral vector LXSN116E6E7 containing the human papilloma virus oncogenes 16 E6 and E7. The transfected equine kidney cell line and equine lung cell line can be propagated for more than 40 passages, whereas the corresponding primary cells only for 10-12 passages. T...
Mifune H, Richter R, Forssmann WG.The distribution of immunoreactivity (IR) for cardiodilatin/atrial natriuretic peptide (CDD/ANP) and brain natriuretic peptide (BNP) was examined immunohistochemically and immuno-electron-microscopically in the equine atrium, using specific antibodies. In the immunohistochemical studies, IR-CDD/ANP and IR-pBNP-26 (porcine BNP-26 immunoreactivity) was detected in the cytoplasm of the auricular cardiocytes, but IR-hBNP-32 (human BNP-32 immunoreactivity) was not. The double immunogold labelling method for IR-hBNP-28 and IR-pBNP-26 revealed that gold particles of different sizes were located in th...
Auer DE, Skelton KV, Tay S, Baldock FC.Total plasma carbon dioxide (TCO2) concentrations were measured in Standardbred horses to determine criteria to discriminate between normal horses and horses with excessive TCO2 concentrations on raceday. TCO2 concentrations from stabled horses were distributed normally with a mean of 30.2 mmol/L and a standard deviation of 1.2 (n = 192) while pre-race TCO2 concentrations were not normally distributed. The results indicate that about 50 horses per million are likely to have TCO2 concentrations greater than or equal to 35 mmol/L and that it is extremely unlikely that a normal horse would have a...
Parsons Aubone AM, Bisiau CM, McCue PM, Bouma GJ.Mammals have a circadian rhythm that is synchronized by a master clock located in the hypothalamic suprachiasmatic nucleus (SCN). The SCN regulates additional clocks located in peripheral tissues, including some involved in endocrine or reproductive functions. Studies in humans and mice report that molecular clocks also exist in the placenta. However, little is known about the presence of "Clock genes," namely Circadian Locomotor Output Cycles Kaput (CLOCK), Brain and Muscle Arnt-Like 1 (BMAL1), Period 1 (PER1), Period 2 (PER2), Cryptochrome 1 (CRY1), and Cryptochrome 2 (CRY2), in equine place...
Sukmawinata E, Sato W, Uemura R, Kanda T, Kusano K, Kambayashi Y, Sato T, Ishikawa Y, Toya R, Sueyoshi M.In this study, the occurrence of antimicrobial-resistant (AMR) enterococci was evaluated in Thoroughbred (TB) racehorses in Japan. Fecal samples were collected from 212 healthy TB racehorses at the Miho and Ritto Training Centers of the Japan Racing Association from March 2017 to August 2018. Isolation and identification were performed by enterococcus selective medium and confirmed to the species using MALDI-TOF MS. Enterococcus faecium and E. faecalis isolates were subjected to antimicrobial susceptibility test against 11 antimicrobials by minimum inhibitory concentration based on recommenda...
Shepard KN, Haffner JC, Neal DL, Grubbs ST, Pearce GL.Plasma adrenocorticotropic hormone (ACTH) concentration is used in the diagnosis of pituitary pars intermedia dysfunction (PPID) in horses. We enrolled 10 horses, 5 PPID-positive and 5 PPID-negative, in our study, September 20-22, 2016. On day 0, 5 mL of whole blood was collected into each of 6 EDTA tubes and immediately placed in a refrigerator at 7°C. One tube was centrifuged within 15 min of collection, followed by centrifugation of one tube from each horse at 4, 8, 12, 24, and 36 h following collection. At each time, centrifuged plasma was pipetted into 1.5-mL polypropylene tubes and stor...
Shang S, Zhang M, Zhao Y, Dang W, Hua P, Zhang S, Wang Z.Due to the thriving development of the modern horse industry and the occurrence of horse related crimes, the demand for methods of individual horse identification, parentage tests and other genetic analyses is increasing. Previous methods had disadvantages that decreased the accuracy of the results, lacked the inclusion of all commonly used short tandem repeats (STR) or increased the experimental cost and time. Objective: We aimed to develop a novel 13-plex STR typing system to resolve the above issues. Methods: Experimental study. Methods: Twelve autosomal and most commonly used di-nucleotide...
Henderson LL, Warren JC.Estradiol 17 alpha-dehydrogenase (EC 1.1.1.148) and estradiol 17 beta-dehydrogenase (EC 1.1.1.62) from horse placenta have been purified to homogeneity. Both enzymes are localized in the microsomal fraction and are solubilized in 1.5% sodium cholate. The 17 alpha- and 17 beta-dehydrogenases are separated by selective elution from hydroxylapatite with 0.5 and 1.0 M potassium phosphate, respectively. Subsequent purification is achieved by two affinity-absorption steps using reactive blue 2-agarose and estriol hemisuccinate-Sepharose. Homogeneous estradiol 17 alpha-dehydrogenase has a specific ac...
Kwok WH, Leung DK, Leung GN, Tang FP, Wan TS, Wong CH, Wong JK.In September 2005, our laboratory detected the presence of 4-androstene-3,17-dione and androsterone in a standard steroid screen of a post-race gelding urine sample received from an overseas authority. All other urine samples from the same batch tested negative. Subsequent gas chromatography/mass spectrometry (GC/MS) confirmatory analyses, however, repeatedly failed to detect any amount of 4-androstene-3,17-dione and androsterone in the suspicious sample. On the other hand, identical results were obtained when the initial GC/MS screening method was repeated on the suspicious sample as well as ...
Al-Ghamdi GM, Kapur V, Ames TR, Timoney JF, Love DN, Mellencamp MA.To determine whether repetitive sequence-based polymerase chain reaction (rep-PCR) could be used to differentiate Streptococcus equi isolates, to examine S equi isolates from throughout the world, and to determine whether a horse had > 1 subtype of S equi during an outbreak of disease. Methods: An initial group of 32 S equi isolates, 63 S equi isolates from various geographic areas, and 17 S equi isolates obtained during outbreaks of disease. Methods: An aliquot of S equi genomic DNA was amplified, using enterobacterial repetitive intergenic consensus primers. Gel electrophoresis was perfor...
Len JA, Beehan DP, Lyle SK, Eilts BE.It was hypothesized that optimal sperm recovery rate (RR) without damage to the sperm would be obtained after centrifugation without a cushion solution. Semen collected three times from six light breed stallions was extended to 25 × 10(6) sperm/mL and centrifuged at CON (noncentrifuged), 900NC (no-cushion), 900C (cushion), 1800NC, and 1800C × g for 10 minutes. Sperm concentration, motility (TM and PM), and intact plasma membranes (PLM) and acrosomes (ACR) pre- and postcentrifugation (D0) and after 24 hours (D1) of cooling were evaluated. The RR in the CON (100 ± 0.0), 900NC (93.7 ± 2.9), a...
Goodrich EL, Behling-Kelly E.The clinical evaluation of lipid metabolism in equids is often limited to the measurement of total cholesterol and triglyceride concentrations. This provides a limited picture of metabolic state and general health, given the continuous exchange of lipid species between various lipoproteins. Major lipoprotein classes in equids include high-density lipoprotein (HDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), and chylomicrons (CM). Unlike large breed horses, donkeys are highly susceptible to hepatic lipidosis. Currently, serum trig...
Consuegra C, Crespo F, Dorado J, Diaz-Jimenez M, Pereira B, Hidalgo M.Lipids and proteins can be used for sperm vitrification to preserve the integrity of sperm membranes or to increase the viscosity of the medium. This study evaluated the effect of low-density lipoproteins (LDL) and milk serum proteins (Pronexcell) for stallion sperm vitrification. Hippex extender (Barex Biochemical Products, The Netherlands), plus 1% of bovine serum albumin and 100 mM of trehalose, was used as control for sperm vitrification. In experiment 1, different concentrations of LDL (L1 = 0.25, L2 = 0.5, L3 = 1%) and in experiment 2 of Pronexcell (P1 = 1, P2 = 5, P3 = 10%)...
Mathes RL, Reber AJ, Hurley DJ, Dietrich UM.To evaluate the effects of topical antifungal drugs and delivery vehicles on the morphology and proliferation rate of cultured equine keratocytes. Methods: 16 corneas obtained from 8 apparently ophthalmologically normal horses < 0.5 hours after euthanasia for reasons unrelated to the study. Methods: Primary cultures of equine keratocytes were obtained from corneal stroma and were exposed to several concentrations of 3 commonly used, topically applied antifungals: natamycin, itraconazole, and miconazole. In addition, effects of drug delivery vehicles DMSO, benzalkonium chloride, and carboxym...
Jouvel C, Maciejewski P, Garcia P, Bonnaire Y, Horning S, Popot MA.A method for the detection of diazepam in horse hair samples by low resolution gas chromatography-mass spectrometry (GC-MS) was developed. Two other techniques, gas chromatography-high-resolution mass spectrometry (GC-HRMS) and high-performance liquid chromatography-atmospheric pressure chemical-ionisation mass spectrometry (HPLC-APCI-MS-MS) were applied on some selected samples. Sample preparation was performed according to a technique previously described for human hair, involving incubation with Sorensen buffer and solvent extraction. Hair samples from different sites such as coat on the ne...
Kawcak CE, Trotter GW, Frisbie DD, McIlwraith CW.To evaluate the effects of a commercially defined, serum-free medium additive on equine articular cartilage explants, compared with effects of serum-free and serum-supplemented media. Methods: Articular cartilage from a 3-year-old, mixed breed horse euthanatized for reasons other than musculoskeletal disease or sepsis. Methods: Media were changed every 48 hours, and the glycosaminoglycan (GAG) content was determined in media collected at each time point. Glycosaminoglycan synthesis by explant chondrocytes, and residual GAG content of articular cartilage (as a measure of explant GAG loss) were ...
Silva ESM, Vaz IS, Rodrigues TS, Brandão FZ, Oquendo PS, Oquendo FMG, Beletti ME, Cuervo-Arango J.Uterocalin (P19) is a lipocalin protein that has binding activity for the provisioning of the embryo with lipids, including those essential for morphogenesis and pattern formation. Therefore, it is thought that P19 plays an important role in supporting the survival of the early embryo. Previous research has shown that the endometrium from acyclic mares expressed more P19 following a simulated long estrus: treatment of 8 mg of estradiol benzoate (EB) split over five consecutive days, followed by long-acting progesterone administration (LA-P4) 72 hours later, when compared to a single 2.5 mg EB ...
