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.
Slusarewicz P, Pagano S, Mills C, Popa G, Chow KM, Mendenhall M, Rodgers DW, Nielsen MK.Intestinal parasites are a concern in veterinary medicine worldwide and for human health in the developing world. Infections are identified by microscopic visualisation of parasite eggs in faeces, which is time-consuming, requires technical expertise and is impractical for use on-site. For these reasons, recommendations for parasite surveillance are not widely adopted and parasite control is based on administration of rote prophylactic treatments with anthelmintic drugs. This approach is known to promote anthelmintic resistance, so there is a pronounced need for a convenient egg counting assay...
Dunbar LK, Mielnicki KA, Dembek KA, Toribio RE, Burns TA.Several tests have been evaluated in horses for quantifying insulin dysregulation to support a diagnosis of equine metabolic syndrome. Comparing the performance of these tests in the same horses will provide clarification of their accuracy in the diagnosis of equine insulin dysregulation. Objective: The aim of this study was to evaluate the agreement between basal serum insulin concentrations (BIC), the oral sugar test (OST), the combined glucose-insulin test (CGIT), and the frequently sampled insulin-modified intravenous glucose tolerance test (FSIGTT). Methods: Twelve healthy, light-breed ho...
Erol E, Jackson C, Horohov D, Locke S, Smith J, Carter C.Determination of fetal serum amyloid A (SAA) concentrations in aborted fetuses can provide valuable information regarding the infectious and/or inflammatory process of abortion in horses. To investigate the relationship between fetal SAA levels and the infectious/inflammatory disease process in feto-placental tissues, a SAA ELISA was used to test heart serum samples of 89 equine fetuses that were necropsied and diagnosed in the following groups: a multiorgan disease process diagnosed with an identified microorganism (14 cases, group 1); only placentitis diagnosed with an identified microorgan...
Kwok WH, Choi TL, So PK, Yao ZP, Wan TS.Xenon can activate the hypoxia-inducible factors (HIFs). As such, it has been allegedly used in human sports for increasing erythropoiesis. Krypton, another noble gas with reported narcosis effect, can also be expected to be a potential and less expensive erythropoiesis stimulating agent. This has raised concern about the misuse of noble gases as doping agents in equine sports. The aim of the present study is to establish a method for the simultaneous detection of xenon and krypton in equine plasma for the purpose of doping control. Xenon- or krypton-fortified equine plasma samples were prepar...
Espina M, Jülke H, Brehm W, Ribitsch I, Winter K, Delling U.Background. Mesenchymal stromal cells (MSCs) are increasingly used for clinical applications in equine patients. For MSC isolation and expansion, a laboratory step is mandatory, after which the cells are sent back to the attending veterinarian. Preserving the biological properties of MSCs during this transport is paramount. The goal of the study was to compare transport-related parameters (transport container, media, temperature, time, cell concentration) that potentially influence characteristics of culture expanded equine MSCs. Methods. The study was arranged in three parts comparing (I) fiv...
Wang Y, Mao Q, Chang H, Wu Y, Pan S, Li Y, Zhang Y.Integrins can function as receptors for foot-and-mouth disease virus (FMDV) in epithelium. Horses are believed to be insusceptible to this disease, but the mechanism of resistance remains unclear. To detect whether FMDV can use integrin to attach to equine epithelial, we compared the utilities of αvβ3 and αvβ6 between bovine and equine kidney epithelial cells (KECs). Equine KECs showed almost equal efficiency to those of bovine. Further, the integrin αv, β3, and β6 subunits from bovine and equine were cloned and vectors were transfected into SW480 cells and COS-1 cells alone or together...
Chindera K, Mahato M, Sharma AK, Horsley H, Kloc-Muniak K, Kamaruzzaman NF, Kumar S, McFarlane A, Stach J, Bentin T, Good L.To combat infection and antimicrobial resistance, it is helpful to elucidate drug mechanism(s) of action. Here we examined how the widely used antimicrobial polyhexamethylene biguanide (PHMB) kills bacteria selectively over host cells. Contrary to the accepted model of microbial membrane disruption by PHMB, we observed cell entry into a range of bacterial species, and treated bacteria displayed cell division arrest and chromosome condensation, suggesting DNA binding as an alternative antimicrobial mechanism. A DNA-level mechanism was confirmed by observations that PHMB formed nanoparticles whe...
McElhone RL, Meakin GE, French JC, Alexander T, Morgan RM.A study was designed to investigate the effects of external variables, including blood type, flooring surface, footwear tread depth and blood dryness, on the appearance of blood-based footwear marks, with particular reference to simulating a specific casework scenario. Results showed that footwear marks left in human blood tended to be of greater quality than those in equine blood, highlighting a potential issue in applying data generated with equine blood to human bloodstains in casework. Footwear tread effects were also dependent on blood type, but the type of flooring surface did not affect...
Hue ES, Fortier CI, Laurent AM, Quesnelle YF, Fortier GD, Legrand LJ, Pronost SL.The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 norm. After the development and first validation step, two distinct characterization steps were performed according to the AFNOR norm: (a) characterization of the qRT-PCR assay alone and (b) characterization of the whole analytical method. The validation of the whole analytical method included the portrayal of all steps between the extraction of nucleic acids and the final PCR analysis. Validation of the whole method is very important for vi...
Duan H, Gao JF, Su X, Fu X, Yue DM, Gao Y, Qiu JH, Wang CR.Triodontophorus spp. parasitizes the large intestine of equine, causing strongylid diseases. The present study assessed genetic variation in five gene regions within and between Triodontophorus brevicauda and Triodontophorus nipponicus from Heilongjiang Province and the Inner Mongolia Autonomous region. The five gene markers were three mitochondrial (mt) genes, cytochrome c oxidase subunit I (cox1), NADH dehydrogenase subunit 5 (nad5), cytochrome b (cytb); and two ribosomal RNA genes, the internal transcribed spacer 1 (ITS1) and the internal transcribed spacer 2 (ITS2). Partial (p) sequences o...
Crawley JA, Chapman SN, Lummaa V, Lynsdale CL.Parasite infection status, intensity and resistance have traditionally been quantified via flotation techniques, but the need for immediate analysis following defecation imposes limitations and has led to the use of several faecal storage techniques. However, their effect on nematode egg counts has not been systematically validated and is often generalised across taxa despite evidence of differences between species. Here, we take the domestic horse Equus ferus caballus as a model to examine the impact of commonly used storage techniques on egg recovery: 1) high and low concentrations of ethano...
Van de Water E, Oosterlinck M, Duchateau L, Pille F.The purpose of this study was to determine whether the scil Vet abc Plus(+) (SCIL Animal Care Company, Altorf, France), an impedance hematology analyzer, can accurately quantify and differentiate nucleated blood cells (NBCs) in equine synovial fluid. Synovial fluid samples (n=242) in different stages of experimentally induced inflammation were analyzed with and without hyaluronidase pretreatment and compared to manual hemocytometer counts and smear reviews. No significant effect of hyaluronidase pretreatment was observed. Total nucleated cell counts of the scil Vet abc Plus(+) were significant...
Hansson A, Knych H, Stanley S, Thevis M, Bondesson U, Hedeland M.Selective androgen receptor modulators (SARMs) are prohibited in sports due to their performance enhancing ability. It is important to investigate the metabolism to determine appropriate targets for doping control. This is the first study where the equine metabolites of SARMs S1, S4 (Andarine) and S22 (Ostarine) have been studied in plasma. Methods: Each SARM was administered to three horses as an intravenous bolus dose and plasma samples were collected. The samples were pretreated with protein precipitation using cold acetonitrile before separation by liquid chromatography. The mass spectrome...
Sander J, Cavalleri JM, Terhardt M, Bochnia M, Zeyner A, Zuraw A, Sander S, Peter M, Janzen N.Hypoglycin A (2-amino-3-(2-methylidenecyclopropyl)propanoic acid) is the plant toxin shown to cause atypical myopathy in horses. It is converted in vivo to methylenecyclopropyl acetic acid, which is transformed to a coenzyme A ester that subsequently blocks beta oxidation of fatty acids. Methylenecyclopropyl acetic acid is also conjugated with carnitine and glycine. Acute atypical myopathy may be diagnosed by quantifying the conjugates of methylenecyclopropyl acetic acid plus a selection of acyl conjugates in urine and serum. We describe a new mass spectrometric method for sample volumes of <0...
Nakayama SM, Ikenaka Y, Hayami A, Mizukawa H, Darwish WS, Watanabe KP, Kawai YK, Ishizuka M.Research on drug metabolism and pharmacokinetics in large animal species including the horse is scarce because of the challenges in conducting in vivo studies. The metabolic reactions catalyzed by cytochrome P450s (CYPs) are central to drug pharmacokinetics. This study elucidated the characteristics of equine CYPs using diazepam (DZP) as a model compound as this drug is widely used as an anesthetic and sedative in horses, and is principally metabolized by CYPs. Diazepam metabolic activities were measured in vitro using horse and rat liver microsomes to clarify the species differences in enzy...
Tangtrongsup S, Kisiday JD.Dexamethasone is known to support mesenchymal stem cell (MSC) chondrogenesis, although the effects of dose and timing of exposure are not well understood. The objective of this study was to investigate these variables using a laboratory model of MSC chondrogenesis. Methods: Equine MSCs were encapsulated in agarose and cultured in chondrogenic medium with 1 or 100 nM dexamethasone, or without dexamethasone, for 15 days. Samples were analyzed for extracellular matrix (ECM) accumulation, prostaglandin E2 and alkaline phosphatase secretion, and gene expression of selected collagens and catabolic e...
Kinsley R, Scott SD, Daly JM.Serological assays provide an indirect route for the recognition of infectious agents via the detection of antibodies against the infectious agent of interest within serum. Serological assays for equine influenza A virus can be applied for different purposes: diagnosing infections; subtyping isolates; surveillance of circulating strains; and to evaluate the efficacy of vaccines before they reach the market. Haemagglutination inhibition (HI) and single radial haemolysis (SRH) assays are most commonly used in the equine field. This review outlines how both these assays together with virus neutra...
Carslake H, Karikoski N, Pinchbeck G, McGowan C.Serum insulin concentration is commonly measured during investigation of suspected endocrinopathic disease in horses, but immediate analysis is frequently unavailable. The aim of this study was to examine the effect of storing samples at room temperature for 72 h as serum and as whole blood, compared to immediate separation and freezing. Samples from 14 horses were evaluated. Correlation was excellent for all comparisons (≥0.992). Bland-Altman plots revealed a negative bias (mean difference 2.16 µIU/mL) in samples stored as whole blood compared to serum, but this difference was not cons...
Kumar CS, Sivaramakrishna D, Ravi SK, Swamy MJ.Seminal fibronectin type-II (Fn-II) proteins interact with choline phospholipids present on the sperm plasma membrane and play a crucial role in sperm capacitation. Crystal structure of phosphorylcholine (PrC) complex of PDC-109, the major bovine Fn-II protein, together with fluorescence spectroscopic studies has shown that tryptophan residues are crucial for its specific interaction with choline phospholipids. In the present study, the heterogeneity and microenvironment of tryptophan residues in HSP-1/2, a major protein of horse seminal plasma (which is homologous to PDC-109) were investigate...
Oldenhof H, Schütze S, Wolkers WF, Sieme H.Sperm chromatin structure and condensation determine accessibility for damage, and hence success of fertilization and development. The aim of this study was to reveal characteristic spectral features coinciding with abnormal sperm chromatin packing (i.e., DNA-protein interactions) and decreased fertility, using Fourier transform infrared spectroscopy. Chromatin structure in spermatozoa obtained from different stallions was investigated. Furthermore, spermatozoa were exposed to oxidative stress, or treated with thiol-oxidizing and disulfide-reducing agents, to alter chromatin structure and pack...
Roach J, Schnobrich M, Ellerbrock R, Feijo L, Bradecamp E, Alvarenga MA, Kline K, Canisso I.This study compares two methods for seminal plasma removal by evaluating sperm recovery rates, and motility and morphology of cooled-stored semen. Ejaculates were divided into three groups: control, filtration and cushioned centrifugation. Semen was extended to 25 million sperm/ml using a skim-milk-based extender and stored at 5°C for all groups. Sperm motility (total motility (%TM) and progressive motility (%PM)) was determined at 0, 24, 48 and 72 hours by a computer-assisted sperm analyser. Sperm morphology was assessed using differential interference microscopy. Overall, %TM of the cen...
Handing KB, Shabalin IG, Szlachta K, Majorek KA, Minor W.Serum albumin (SA) is the main transporter of drugs in mammalian blood plasma. Here, we report the first crystal structure of equine serum albumin (ESA) in complex with antihistamine drug cetirizine at a resolution of 2.1Å. Cetirizine is bound in two sites--a novel drug binding site (CBS1) and the fatty acid binding site 6 (CBS2). Both sites differ from those that have been proposed in multiple reports based on equilibrium dialysis and fluorescence studies for mammalian albumins as cetirizine binding sites. We show that the residues forming the binding pockets in ESA are highly conserved in h...
Cleaver N, Parikh K, Kazlouskaya V, Elston D.Equine type melanoma can mimic deep penetrating nevus (DPN), making histologic diagnosis challenging. We sought to investigate if the pattern of collagen polarization could be helpful in this setting. A total of 52 specimens were reviewed with polarized microscopy to determine whether refractile collagen was present within melanocytic nests vs. surrounding but not within the nests. Seven of eight (87.5%) equine type melanomas demonstrated refractile collagen within melanocytic nests in part or all of the lesion. In contrast, DPN showed no refractile collagen within the melanocytic nests. Inste...
Matta C, Zhang X, Liddell S, Smith JR, Mobasheri A.There is insufficient knowledge about the chondrocyte membranome and its molecular composition. Objective: To develop a Triton X-114 based separation technique using nanoLC-MS/MS combined with shotgun proteomics to identify chondrocyte membrane proteins. Methods: Articular chondrocytes from equine metacarpophalangeal joints were separated into hydrophobic and hydrophilic fractions; trypsin-digested proteins were analysed by nanoLC-MS/MS. Results: A total of 315 proteins were identified. The phase extraction method yielded a high proportion of membrane proteins (56%) including CD276, S100-A6 an...
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...
Blumer R, Maurer-Gesek B, Gesslbauer B, Blumer M, Pechriggl E, Davis-López de Carrizosa MA, Horn AK, May PJ, Streicher J, de la Cruz RR, Pastor ÁM.To test whether palisade endings are a general feature of mammalian extraocular muscles (EOMs). Methods: Thirteen species, some frontal-eyed (human, monkey, cat, and ferret), and others lateral-eyed (pig, sheep, calf, horse, rabbit, rat, mouse, gerbil, and guinea pig) were analyzed. Palisade endings were labeled by using different combinations of immunofluorescence techniques. Three-dimensional reconstructions of immunolabeled palisade endings were done. Results: In all frontal-eyed species, palisade endings were a consistent feature in the rectus EOMs. Their total number was high and they exh...
Yamanaka T, Nemoto M, Bannai H, Tsujimura K, Kondo T, Matsumura T, Gildea S, Cullinane A.Equine influenza (EI) is a highly contagious disease caused by viruses of the H3N8 subtype. The rapid diagnosis of EI is essential to reduce the disease spread. Many rapid antigen detection (RAD) tests for diagnosing human influenza are available, but their ability to diagnose EI has not been systematically evaluated. Objective: The aim of this study was to compare the performance of 22 RAD tests in the diagnosis of EI. Methods: The 22 RAD tests were performed on fivefold serial dilutions of EI virus to determine their detection limits. The four most sensitive RAD tests (ImmunoAce Flu, BD Flu ...
Stasiak K, Rola J, Zmudzinski JF.A highly sensitive and specific real-time PCR assay was used for detection and quantitation of equine herpesvirus type 1 (EHV-1) in the different internal organs of aborted fetuses. Tissue samples from 23 aborted fetuses submitted to the Department of Virology of the National Veterinary Research Institute in Pulawy between 2012 and 2013 were used for testing. Total DNA was extracted using a phenol-chloroform-isoamyl alcohol standard protocol. A real-time PCR with forward and reverse primers encompassing a highly conserved region encoding viral glycoprotein B was adapted for diagnosis of EHV-1 ...
Olaciregui M, Luño V, Martí JI, Aramayona J, Gil L.During the freeze-drying procedure, sperm DNA might become damaged by both freezing and drying stresses. Sperm DNA status can be detected using well-established assays; however, most techniques are expensive and involve elaborate protocols and equipment. Indirect assessments can provide alternative strategies. The objective of this study was to compare a simple test of DNA status using Diff-Quik (DQ) with two established procedures: acridine orange test (AOT) and sperm chromatin dispersion (SCD) on freeze-dried (FD) stallion spermatozoa. Ejaculated spermatozoa from three stallions were freeze-...
Pusterla N, Magdesian KG, Mapes SM, Zavodovskaya R, Kass PH.The ante mortem diagnosis of equine multinodular pulmonary fibrosis (EMPF) relies on histopathological results and polymerase chain reaction (PCR)-positive equine herpesvirus (EHV)-5 testing of lung tissue. Polymerase chain reaction detection of EHV-5 in bronchoalveolar lavage fluid (BALF) is commonly used to support a diagnosis of EMPF. However, the diagnostic power of EHV-5 testing on BALF and other biological samples such as blood and nasal secretions has yet to be shown to support a diagnosis of EMPF. Objective: To determine the frequency of detection and the viral loads of EHV-5 by quanti...
Sjostrom L, al-Abdulla IH, Rawat S, Smith DC, Landon J.Commercial antivenoms produced in horses were compared with monospecific antivenoms raised in sheep against Crotalus durissus terrificus, Crotalus atrox, Crotalus adamanteus, Micrurus fulvius fulvius, Naja naja, Naja kaouthia, Echis ocellatus, Vipera lebetina deserti, Vipera berus berus and Vipera ammodytes ammodytes venom. Antibodies raised by immunizing sheep with C. d. terrificus venom were more effective than their equine counterparts in preventing lethal toxicity in mice (ED50), in inhibiting the venom's pharmacological effects (haemolysis, platelet aggregation and coagulation), and in ne...
Martoriati A, Caillaud M, Goudet G, Gérard N.Interleukin 1 beta (IL-1 beta) inhibits the LH-induced resumption of meiosis of equine oocytes in vitro. The present study was performed to clarify this inhibitory effect of IL-1 beta by testing increasing concentrations of IL-1 beta, and by measuring the effect of addition of IL-1 receptor antagonist (IL-1RA) to the culture medium. The effect of IL-1 beta on epidermal growth factor (EGF)-induced resumption of meiosis was also studied. Cumulus-oocyte complexes (COCs) were collected from subordinate follicles on ovaries obtained from an abattoir. In five distinct experiments, COCs were cultured...
Bełżecki G, Miltko R, Michałowski T, McEwan NR.This paper describes cultivation methods for ciliates from the digestive tract of horses. Members of three different genera were successfully grown in vitro for short periods of time. However, only cells belonging to the genus Blepharocorys, which resides in the horse's large intestine, were maintained for longer periods. This Blepharocorys culture was successfully grown in vitro after inoculation of freshly excreted horse faeces in culture medium containing a population of bacteria. The ciliates survived for over six months, and the density of their population varied between 1.7 × 10(3) and ...
Epstein KL, Brainard BM, Lopes MA, Barton MH, Moore JN.To develop a standardized technique for thrombelastography (TEG) analysis in healthy adult horses, with and without the ex vivo addition of tissue factor (TF) as an activator. To determine reference intervals for TEG parameters in the horse, and to determine if traditional coagulation tests correlate with TEG. Methods: Prospective, observational. Methods: Veterinary teaching hospital. Methods: Twenty-six healthy adult horses. Methods: None. Results: Thrombelastography with (TF-TEG) and without (TEG) the addition of TF performed by 4 operators. Coagulation profiles (prothrombin time, activated ...
Jiménez-Aristazábal RF, Carmona JU, Prades M.The aims of the present study were (1) to describe the microscopic and ultrastructural appearance of equine platelet-rich fibrin (PRF) clots and (2) to determine the release and degradation of transforming growth factor beta 1 (TGF-β) and insulin-like growth factor type I (IGF-I) from PRF clots incubated over 14 days. Whole blood from six horses was collected into plain tubes and centrifuged at 240 g for 8 minutes. Clots were evaluated by histology and by both transmission and scanning electronic microscopy (TEM and SEM). Growth factor concentrations were measured by ELISA at 48-hour inter...
Sweeney CR, Russell GE.To examine total protein concentration and cell counts of sequentially collected samples of CSF to determine whether blood contamination decreases in subsequent samples and whether formulas used to correct nucleated cell count and total protein concentration are accurate. Methods: Case series. Methods: 22 horses. Methods: For each horse, 3 or 4 sequential 2-ml samples of CSF were collected from the subarachnoid space in the lumbosacral region into separate syringes, and blood was obtained from the jugular vein. Total protein concentration, nucleated cell count, and RBC counts were determined i...
Marth CD, Firestone SM, Hanlon D, Glenton LY, Browning GF, Young ND, Krekeler N.Persistent mating-induced endometritis (PMIE) severely decreases fertility in horses. The aim of the present study was to evaluate differences between horses susceptible to PMIE and a control group in terms of the expression of selected immune response and effector genes, and the effects of oestrous cycle stage on this expression. Endometrial biopsies from 18 uterine samples of mares in the control group (eight in dioestrus, 10 in oestrus) and 16 PMIE-susceptible mares (four in dioestrus, 12 in oestrus) were analysed by quantitative real-time reverse transcription-polymerase chain reaction. Ge...
Dunnett M, Harris RC, Orme CE.A simple, robust and reproducible analytical method for the determination of phosphocreatine (PCr), creatine (Cr) and creatinine (Cn) in equine skeletal muscle is presented. The technique used isocratic reverse-phase ion-pairing high-performance liquid chromatography. Neutralized perchloric acid extracts of equine muscle biopsies were analysed and the values obtained were compared with determinations from an established enzymic procedure. Good resolution of all three metabolites was achieved within a retention time of less than 11 min. Linearity for each metabolite within the concentration ran...
Bramer SA, Macedo A, Klein C.Secretion of histotroph during the prolonged pre-implantation phase in mares is crucial to pregnancy maintenance, manifested as increased embryonic loss in mares with age-related endometrial degeneration. Glycogen content of uterine histotroph is higher during the progesterone-dominated phase of the estrous cycle in mares, but regulatory mechanisms are not well understood. Methods: mRNA expression of glycogen-metabolizing enzymes (HK1, HK2, GSK3B, GYS1, PEPCK, PKM, PYGM) in endometrial samples were compared among mares in anestrus, estrus, and at Day 12 of diestrus and pregnancy. In addition, ...
Lavoie JP, Lefebvre-Lavoie J, Leclere M, Lavoie-Lamoureux A, Chamberland A, Laprise C, Lussier J.Gene expression analyses are used to investigate signaling pathways involved in diseases. In asthma, they have been primarily derived from the analysis of bronchial biopsies harvested from mild to moderate asthmatic subjects and controls. Due to ethical considerations, there is currently limited information on the transcriptome profile of the peripheral lung tissues in asthma. Objective: To identify genes contributing to chronic inflammation and remodeling in the peripheral lung tissue of horses with heaves, a naturally occurring asthma-like condition. Methods: Eleven adult horses (6 heaves-af...
González Medina S, Hyde C, Lovera I, Piercy RJ.Hypoglycin A (HGA) toxicity, following ingestion of material from certain plants, is linked to an acquired multiple acyl-CoA dehydrogenase deficiency known as atypical myopathy, a commonly fatal form of equine rhabdomyolysis seen worldwide. Whilst some plants are known to contain this toxin, little is known about its function or the mechanisms that lead to varied HGA concentrations between plants. Consequently, reliable tools to detect this amino acid in plant samples are needed. Analytical methods for HGA detection have previously been validated for the food industry, however, these technique...
Marsh AE, Barr BC, Madigan J, Lakritz J, Conrad PA.To identify Sarcocystis neurona-specific DNA sequences in the nuclear small subunit ribosomal RNA (nss-rRNA) gene that could be used to distinguish S neurona from other closely related protozoal parasites, and to evaluate a polymerase chain reaction (PCR) test, using broad based primers and a unique species-specific probe on CSF for detection of S neurona in equids. Methods: Sequencing of the nuclear small subunit ribosomal RNA gene from a new S neurona isolate (UCD 1) was performed. The sequence was compared with that of other closely related Sarcocystidae parasites. From this sequence, conse...
Choi YH, Roasa LM, Love CC, Varner DD, Brinsko SP, Hinrichs K.This study was conducted to evaluate in vivo and in vitro development of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection. Oocytes were collected from slaughterhouse-derived ovaries, matured in vitro, and injected with frozen-thawed stallion sperm. In vivo development was assessed after transfer of injected oocytes to the oviducts of recipient mares. Mares were killed 7.5-8.5 days after transfer and the uterus and oviducts flushed for embryo recovery. Of 132 injected oocytes transferred, 69 (52%) were recovered; of these, 25 (36%) were blastocysts with a blastocoe...
Nemoto M, Hata H, Higuchi T, Imagawa H, Yamanaka T, Niwa H, Bannai H, Tsujimura K, Kondo T, Matsumura T.We evaluated antigen detection kits for human rotavirus with regard to their usefulness for diagnosing equine rotavirus infection. Limiting dilution analyses showed that of the seven kits investigated the Dipstick `Eiken' Rota (Dipstick) had the highest sensitivity to two serotypes of equine rotavirus. The Dipstick did not cross-react with several equine intestinal pathogens. An investigation using 249 fecal samples indicated that the sensitivity of the Dipstick was 81.9% and 47.3%, and its specificity was 98.2% and 99.0%, and its concordance rate was 92.8% and 68.3%, compared with values obta...
Artiushin S, Tong Y, Timoney J, Lemieux B, Schlegel A, Kong H.A simple and portable assay for detection of Streptococcus equi subspecies equi has been developed based on amplification of S. equi-specific sequence using a thermophilic helicase-dependent reaction followed by visual detection of the amplicon in a disposable lateral flow cassette. An experimental kit (IsoAmp™ SE) was evaluated. Analytical sensitivity was 50 copies of S. equi genomic DNA per reaction. The IsoAmp SE assay had 100% specificity when applied to nasal swabs and washes. The assay was more sensitive than culture but less sensitive than nested polymerase chain reaction (PCR). The t...
Owens SD, Snipes J, Magdesian KG, Christopher MM.Blood typing before transfusion minimizes the risk of transfusion reactions and prevents immunization of the recipient against incompatible RBC antigens. The major RBC antigens that warrant identification before packed RBC or whole blood transfusions in horses are Ca and Aa. Standard blood-typing protocols are time-consuming (2.5-3.0 hours) and impractical in emergency settings. Objective: The purpose of this study was to determine whether equine RBCs could be typed for Ca and Aa antigens using sera from horses with RBC antibodies in a modified rapid (15 minute) blood-typing protocol. Methods:...
Agius CT, Nagesha HS, Studdert MJ.A new equine herpesvirus, provisionally designated equine herpesvirus 5 (EHV5; Browning and Studdert (1987) J. Gen. Virol. 68, 1441-1447), was examined for the degree of genomic difference from equine herpesvirus 2 (EHV2) by Southern hybridizations. EHV5 and EHV2 whole genomic DNA probes were highly specific for homologous DNA only, indicating that significant genomic difference exists between the two viruses. Restriction endonuclease analysis of EHV5 strain 2-141 (EHV5.2-141) revealed that the genome is 179 kb and exists as a single isomer. Clones representing 82% of the genome were obtained ...
Leeb T, Müller EJ, Roosje P, Welle M.Molecular genetics has made significant advances in the analysis of hereditary dermatoses during the last several years. Objective: To provide an update on currently available genetic tests for skin diseases of dogs, cats and horses, and to aid the veterinary clinician in the appropriate selection and applications of genetic tests. Methods: The scientific literature on the topic was critically reviewed. The list of known causative variants for genodermatoses and hair morphology traits was compiled by searching the Online Mendelian Inheritance in Animals (OMIA) database. Results: Genetic testin...
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...
Grippa E, Santini L, Castellano G, Gatto MT, Leone MG, Saso L.Ethyl acetate extracts of equine serum, containing 0-5 microg/ml of hydrocortisone (HYD), dexamethasone (DEX), oxyphenbutazone (OPB), indomethacin (IND), phenylbutazone (PB) and probenecid as internal standard, were evaporated with nitrogen, resuspended in methanol and analyzed by HPLC, using a C-18 column equilibrated with 51:49 acetonitrile-water, 0.1% trifluoroacetic acid, at 1 ml/min. The eluate was monitored at 254 nm. The selectivity (inter-assay C.V.<4%), sensitivity (limits of quantitation of 0.25 microg/ml for HYD, DEX and IND, 0.5 microg/ml for PB and 1 microg/ml for OPB, despite ...
Lonning SM, Zhang W, Leib SR, McGuire TC.Cytotoxic T lymphocytes (CTL) appear to be critical in resolving or reducing the severity of lentivirus infections. Retroviral vectors expressing the Gag/Pr or SU protein of the lentivirus equine infectious anemia virus (EIAV) were constructed and used to evaluate EIAV-specific CTL responses in horses. Three promoters, cytomegalovirus, simian virus SV40, and Moloney murine sarcoma virus (MoMSV) long terminal repeat (LTR), were used, and there was considerable variation in their ability to direct expression of Gag/Pr and SU. Vectors expressing EIAV proteins under the direction of MoMSV LTR and ...
Bannai H, Nemoto M, Tsujimura K, Yamanaka T, Kondo T, Matsumura T.Virus-neutralizing (VN) testing is essential for evaluating virus-specific immunity in equine herpesvirus type-1 (EHV-1) infection. We developed a focus-reduction neutralization test (FRNT) for EHV-1 using 96-well plates for faster large-scale testing with sufficient sensitivity. We used an overlay medium containing Avicel (FMC Biopolymer), a microcrystalline cellulose with lower viscosity than the methylcellulose. The foci were visualized by immuno-staining with anti-EHV-1 gp14 monoclonal antibody. The FRNT successfully detected seroconversion in horses experimentally infected with EHV-1 (n =...
Magnuson NS, Perryman LE, Wyatt CR, Mason PH, Talmadge JE.Peripheral blood mononuclear cells from foals with hereditary severe combined immunodeficiency (SCID) have morphologic characteristics of large granular lymphocytes (LGL). Attempts to demonstrate cytotoxic activity were without success unless the LGL were incubated with 100 U of human recombinant interleukin 2 (rIL 2)/ml for 24 hr. With rIL 2 incubation, low effector to target ratios (10:1) consistently yielded high levels of cytotoxic activity (30 to 50%) in a standard 4-hr 51Cr-release assay using YAC-1 lymphoma or K562 erythroleukemia cell lines as targets. Monoclonal antibody EqT12 reacted...
Henson FM, Davenport C, Butler L, Moran I, Shingleton WD, Jeffcott LB, Schofield PN.The effects of insulin and insulin-like growth factors (IGFs) I and II on fetal and foal chondrocytes were investigated in vitro. Chondrocytes from the lateral trochlear ridge of the distal femur were obtained from 2 fetuses (280 and 320 days gestation) and one 4-day-old foal and cultured. Membrane proteins consistent with type 1 and type 2 IGF receptors were demonstrated by radioligand cross linking and equilibrium binding analysis. It was demonstrated that both IGF-I and IGF-II acted as mitogens for isolated equine chondrocytes when present as the sole mitogenic factor in monolayer culture. ...
Pickles K, Pirie RS, Rhind S, Dixon PM, McGorum BC.Bronchoalveolar lavage fluid (BALF) samples are often subject to time delays, possibly with temperature fluctuations, between collection and processing. The aim of this study was to evaluate the effects of time, temperature and 2 different fixatives on equine BALF cytology, in order to develop guidelines for optimal equine BALF storage conditions. Total nucleated cell count (TCC), differential cell counts (DCC), absolute cell counts (ACC), cell viability, cell morphology and bacterial growth of BALF samples stored at 4, 18 (+/- addition of formalin- or alcohol-based fixatives) and 38 degrees C...
Sandbaumhüter FA, Thormann W.Glucuronidation catalyzed by uridine-5'-diphospho-glucuronosyl-transferases (UGTs) is the most important reaction in phase II metabolism of drugs and other compounds. O-glucuronidation is more common than N-glucuronidation. The anesthetic, analgesic and antidepressive drug ketamine is metabolized in phase I by cytochrome P450 enzymes to norketamine, hydroxynorketamine (HNK) diastereomers and dehydronorketamine (DHNK). Equine urine samples collected two hours after ketamine injection were treated with β-glucuronidase and analyzed with three enantioselective capillary electrophoresis assays. Co...
Fjeldsgaard BE, Paulsen BS.Extracts of horse dander (HD) and horse hair and skin scrapings (HHSS) have been compared with respect to their content of proteins and carbohydrates. The protein content of HD is more than double that of HHSS, while the carbohydrate content is of the same order. SDS-PAGE and IEF, both combined with immunoblotting, and CIE/CRIE showed the IgE-binding ability of the proteins/glycoproteins present in the two extracts. SDS-PAGE/immunoblotting showed the presence of mainly the same IgE-binding bands in the two extracts. Nine were detected in HD, and seven in HHSS. Four of these were glycoproteins....
Goudet G, Belin F, Bézard J, Gérard N.In the equine species, a large proportion of oocytes fail to complete meiosis during in-vitro culture. The biochemical and molecular basis of this failure is unknown. The meiotic cell cycle is controlled in part by the maturation-promoting factor (MPF) and the mitogen-activated protein kinase (MAPK). In this study, we evaluated the oocyte competence for in-vitro maturation and the expression of MPF components (p34cdc2 and cyclin B) and MAPK after in-vitro culture. The maturation rate was influenced by the culture medium and the physiological stage of the mare at the time of oocyte recovery. We...
Caron JP, Gandy JC, Schmidt M, Hauptman JG, Sordillo LM.To compare the effects of triamcinolone acetonide (TA) and methylprednisolone acetate (MPA) on expression of selected chondrocyte genes in recombinant equine interleukin-1β (reIL-1β) stimulated articular cartilage explants. Methods: In vitro experiment. Methods: Horses (n = 6). Methods: Articular cartilage explants from 2- to 3- year-old horses were exposed to reIL-1β in the presence and absence of TA and MPA at 10(-7) and 10(-6) M. Resting levels of mRNA of anabolic and catabolic genes of chondrocyte origin were quantified using qPCR after 6- and 12-hour incubations. Genes of interest incl...
Brown JJ, Thomson W, Clegg P, Eyre S, Kennedy LJ, Matthews J, Carter S, Ollier WE.The full extent of the polymorphism of ELA-DRA in Equidae is not yet known. Given the apparent differences in DRA polymorphisms between Equidae and other species, the aims of this study were to more fully characterize ELA-DRA, determine the extent of gene polymorphism and establish the allele-frequency distribution. An allele reference panel for the second exon of ELA-DRA was established by sequence-based typing of 69 equine DNA samples consisting of various breeds of domestic horse (Equus caballus), together with donkeys (Equus asinus), Grant's zebras (Equus boehmi) and one onager (Equus hemi...
