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.
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...
Peng Z, Shen D, Zhang D, Li X, Wang L, Zhai Q, Hou Z, Li H.The equine Parascaris spp. is large, parasitic nematodes, and predominantly focuses on the intestine of foals and young weanlings. There are two roundworms, Parascaris equorum and Parascaris univalens, recognized among equine hosts. In this study, all fifty-nine Parascaris worms were harvested from three different equine hosts (twenty specimens from Equus zebra, twenty specimens from E. caballus, and nineteen specimens from E. asinus). The ribosomal gene (ITS) and mitochondrial genes (cox1 and nadh1) were amplified to identify and genetically characterize these worms. Analysis of ITS sequences...
The typical vertebrate centromeres contain long stretches of highly repeated DNA sequences (satellite DNA). We previously demonstrated that the karyotypes of the species belonging to the genus are characterized by the presence of satellite-free and satellite-based centromeres and represent a unique biological model for the study of centromere organization and behavior. Using horse primary fibroblasts cultured in vitro, we compared the segregation fidelity of chromosome 11, whose centromere is satellite-free, with that of chromosome 13, which has similar size and a centromere containing long s...
Gonzalez-Castro RA, Trentin JM, Carnevale EM, Graham JK.We examined the effects of different freezing extenders, cryoprotectant agents (CPA) and initial thawing temperatures for preparing doses of refrozen stallion sperm for intracytoplasmic sperm injection (ICSI). Single ejaculates, from twelve stallions, were frozen in lactose-EDTA-egg yolk extender (LE) with 5% glycerol. In experiment 1, sperm were initially thawed to 5 °C or 37 °C, before being diluted in LE or skim milk-egg yolk extender (SMEY) containing either 5% glycerol (GLY), 5% methylformamide (MF) or 5% of a combination of both (GMF). In experiment 2, frozen sperm were initially t...
Viesselmann LC, Flatland B, Stokol T, Sisson S, Schaefer DMW.Accurate erythrocyte measurements with ADVIA hematology analyzers require isovolumetric cell sphering in one reaction and hemolysis in another. However, camelid erythrocytes are resistant to sphering and osmotic lysis, and no published evaluation of ADVIA methods for camelids exists. Objective: The objectives were to demonstrate whether camelid erythrocytes sphere in the ADVIA red blood cell/platelet (RBC/PLT) reagent and lyse in the ADVIA cyanide HGB reagent, and to determine optimal ADVIA settings for camelids. Methods: Camelid and canine blood were diluted to 1:625 in RBC/PLT reagent and ev...
Bonelli F, Laus F, Briganti A, Evangelista F, Bazzano M, Conte G, Sgorbini M.There has been increasing interest in blood gas analysis in donkeys. "Point-of-care (POC) testing" is a diagnostic testing performed on or immediately next to the patient. This study assesses the agreement between two POC blood gas analyzers in donkeys. Arterial and venous blood samples were collected from 17 donkeys and analyzed using a fully automated blood gas analyzer (ABL 700 Series Radiometer, Denmark) (RAD) and two POC blood gas analyzers (i-STAT System; VetStat, Idexx). The parameters revealed by all three devices were submitted to a canonical discriminant analysis, to evaluate which p...
Shakarami MH, Mohammadabadi T, Motamedi H, Sari M, Teimouri Yansari A.This study was conducted to isolate and identify the cellulolytic bacteria from gastrointestinal tract of Arabian horse and investigate their effect on the nutritional value of wheat straw (WS). Results: Fresh faeces were collected from four Arabian horses. The cellulose-hydrolytic bacteria were isolated by using a medium amended with carboxymethyl cellulose (CMC). The activity of CMC was determined by measuring the release of reducing sugars from CMC. Among the isolates, four isolates (L11, L12, L2 and Z2) showed maximum hydrolysis capacity. 16S rRNA sequence analysis showed that these isolat...
Contreras-Aguilar MD, Escribano D, Martínez-Subiela S, Martín-Cuervo M, Lamy E, Tecles F, Cerón JJ.The biochemical components of saliva can change in certain pathologies in horses, for example in acute abdominal disease. The aim of this study was (1) to evaluate if a panel of biochemical analytes usually used in serum can be measured in saliva of horses and (2) to study the possible changes of these biochemical analytes in saliva of horses affected by acute abdominal disease. A panel of 23 analytes was analytically validated in saliva of horses and possible changes in these analytes in a pilot study with six healthy horses and six horses with acute abdominal disease were evaluated. The anal...
Patel T, Magdesian KG, Estell KE, Edman JM, Knych HK.Chloramphenicol is commonly used in horses; however, there are no studies evaluating the pharmacokinetics of veterinary canine-approved tablets. Studies using different formulations and earlier analytical techniques led to concerns over low bioavailability in horses. Safety concerns about human health have led many veterinarians to prescribe compounded formulations that are already in suspension or paste form. The objective of this study was to evaluate the pharmacokinetics of approved chloramphenicol tablets in horses, along with compounded preparations. The hypothesis was that chloramphenico...
McCoy AM, Arrington J, Yau PM.The protein content of amnion is thought to be the primary contributor to its efficacy as a biological dressing for wounds. Protein elution into antibiotic processing media has been reported, but the effect of antiseptic-based processing methods is unknown. Amniotic membranes were collected from eight healthy mares. Samples were collected after removal of gross debris. Tissues were subsequently divided and processed with either 0.05% chlorhexidine or 2% iodine/0.25% acetic acid. After protein extraction and trypsin digestion, the proteins were labeled with 8-plex iTRAQ tags, combined, and anal...
Lerch M, Allbaugh RA, Sebbag L, Mochel JP, Weller P, Borts DJ.Paper spray high-resolution accurate mass spectrometry is a fast and versatile analysis method. This ambient ionization technique enables the quantitation of xenobiotics in complex biological matrices without chromatography or conventional sample extraction. The simplicity, rapidity, and affordability of the paper spray mass spectrometry (PS-MS) method make the technique especially attractive for clinical investigations where fast and affordable sample analysis is crucial. A new PS-MS method for the quantitation of voriconazole in equine tears was developed and validated. For a concentration r...
Griffin RA, Baker M, Aitken RJ, Swegen A, Gibb Z.Stallions experience lower per-cycle conception rates compared to other livestock species, largely because they are selected for breeding based on athletic prowess and not reproductive fitness. Mares are seasonal breeders, and pregnancies cannot be detected until 10-14 days post cover via transrectal ultrasonography. This means the detection of stallion fertility fluctuations is delayed by at least 2 weeks, which within the short breeding season employed by the thoroughbred horse breeding industry, can prove quite costly. For these reasons, there is increased demand for robust laboratory assay...
Consuegra C, Crespo F, Dorado J, Diaz-Jimenez M, Pereira B, Ortiz I, Hidalgo M.Sperm vitrification is a rapid freezing method in which carbohydrates are used as cryoprotectants. The aim of this study was to determine the optimal volume, concentration and type of carbohydrates for stallion sperm vitrification using 0.25 ml straws in comparison to conventional freezing. Ejaculates (n = 54) were collected from six stallions. For vitrification, straws were filled with different volumes (30, 70, 100 μl), sperm concentrations (50, 100, 200 × 10 sperm/ml) and extenders containing sucrose (20, 100, 200 mM), trehalose (50, 100, 200 mM) and raffinose (50, 100, 20...
Casenave P, Leclere M, Beauchamp G, Blais MC.After-hours or out-of-clinic crossmatches are often limited by the lack of access to specialized material and technical expertise. Objective: The goal was to adapt a stall-side crossmatch test for pretransfusion evaluation in horses. Methods: Twelve healthy mares (plasma and blood donors, teaching mares). Methods: In a prospective study, blood from 12 mares was used to compare the results of 132 crossmatches performed with a rapid gel assay to crossmatches performed with a microgel column assay, and with predicted compatibilities based on blood types and detection of antibodies at a reference ...
Canisso IF, Loux S, Scoggin KE, Squires EL, Troedsson MH, Ball BA.Characterisation of fetal fluids in healthy and disease states of pregnant mares can help to unravel the pathophysiology and to identify putative markers of disease. Thus, this study aimed to compare the protein composition of: (1) amniotic and allantoic fluids of healthy mares obtained immediately after euthanasia and (2) allantoic fluid harvested via centesis before and after experimental induction of placentitis via transcervical inoculation of Streptococcus equi ssp zooepidemicus in healthy mares. Fetal fluids were analysed with a high-throughput proteomic technique after in-gel digestion....
Ząbek T, Witarski W, Semik-Gurgul E, Szmatoła T, Kowalska K, Bugno-Poniewierska M.We investigated the activity of chondrogenic markers and variation of methylation patterns in equine cartilaginous cells cultivated in monolayer. The transcriptional and epigenetic effect of the long-term culture of chondrocytes has been evaluated using several passages of chondrocyte cell-lines derived from equine articular cartilage. Using 3 genes as endogenous control we tested the expression of 7 genes important for different stages of chondrocyte differentiation and maturation. CpG islands in RUNX3 locus were inspected for the evaluation of differential methylation state of passaged cell-...
Kinoshita Y, Cloutier AK, Rozak DA, Khan MSR, Niwa H, Uchida-Fujii E, Katayama Y, Tuanyok A.Burkholderia mallei is a Gram-negative bacterium that causes glanders, a zoonotic disease, especially in equine populations (e.g. horses, donkeys, and mules). B. mallei usually grows slowly on most culture media, and this property makes it difficult to isolate from clinical specimens. One of the problems is that B. mallei is easily overgrown by other bacteria, especially in animal specimens collected from non-sterile sites. The aim of this study was to develop a new selective agar for the laboratory diagnosis of glanders. We formulated a new agar, named BM agar, to enrich B. mallei growth, but...
Karimi A, Mosavari N.Burkholderia mallei, the etiologic agent of the disease known as glanders. Clinical and bacteriological diagnosis of glanders is difficult in the early stages of the disease. Currently, mallein (allergic hypersensitivity test) is used for the diagnosis of glanders. The mallein test requires an experienced laboratory person and lasts 48 h. Therefore, in order to quickly diagnose the disease, especially in areas (such as the borders of the country) that cannot be kept animals, new methods should be used to identify the disease. The Rose Bengal is a serological diagnostic test and has been recom...
Waraksa E, Kowalski K, Kłodzińska E, Rola R, Ciekot J, Filipiak W, Bieńkowski T, Namieśnik J.Gamma-oryzanol (GO) has gained special attention in the equine sports industry in recent years due to its touted properties, including the fact that it may cause anabolic effects on muscle growth and reduce fatigue. Many manufactures offer supplements containing GO as a naturally occurring anabolic substance; however, some producers do not declare its presence in product compositions. Taking into consideration the touted properties of GO, its ambiguous effectiveness and the open character of the Prohibited Substances List established by the Fédération Equestre Internationale, there is an urg...
Carossino M, Barrandeguy ME, Erol E, Li Y, Balasuriya UBR.Equine rotavirus A (ERVA) is the leading cause of diarrhea in neonatal foals and has a negative impact on equine breeding enterprises worldwide. Among ERVA strains infecting foals, the genotypes G3P[12] and G14P[12] are the most prevalent, while infections by strains with other genomic arrangements are infrequent. The identification of circulating strains of ERVA is critical for diagnostic and surveillance purposes, as well as to understand their molecular epidemiology. Current genotyping methods available for ERVA and rotaviruses affecting other animal species rely on Sanger sequencing and ar...
Marzano G, Mastrorocco A, Zianni R, Mangiacotti M, Chiaravalle AE, Lacalandra GM, Minervini F, Cardinali A, Macciocca M, Vicenti R, Fabbri R....Di-(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer with endocrine-disrupting properties. In this study, we used an equine model to investigate DEHP concentrations in ovarian follicular fluid (FF), and to determine the effects of exposure of oocytes to potentially toxic concentrations of DEHP during in vitro maturation (IVM) on embryo development and quality. Embryo development was evaluated using time-lapse monitoring (TLM), a photomicroscopic tool that reveals abnormalities in cleavage kinetics unobservable by conventional morphology assessment. Blastocyst bioenergetic/oxidativ...
Desantis S, Accogli G, Albrizio M, Rossi R, Cremonesi F, Lange Consiglio A.Equine amniotic mesenchymal cells (eAMCs) are involved in many mechanisms in tissue regenerative processes. Their secreted vesicles are important effectors in a wide array of biological processes, and contribute to in vivo healing of equine tendon lesions and endometrial inflammation. Glycoconjugates are involved in cellular recognition and in the efficient uptake of extracellular vesicles (EVs) by recipient cells. In this study, we evaluated the surface glycosylation pattern of eAMCs and their EVs from the eAMCs released in conditioned medium. We used a microarray procedure in which eAMCs and...
Szczerba-Turek A, Siemionek J, Ras A, Bancerz-Kisiel A, Platt-Samoraj A, Lipczynska-Ilczuk K, Szweda W.Equine sarcoids are the most common neoplasms in horses. Bovine papilloma- virus type 1 (BPV-1) is the main viral type identified in equine sarcoids in Europe. Objective: The aim of the present study was to genetically evaluate BPV types based on DNA analyses of the CDS of the L1 gene. The presence of BPV DNA was confirmed by Degenerate Oligonucleotide-Primed Polymerase Chain Reaction (DOP PCR) with FAP59/FAP64 consensus primers. Results: The DNA was detected in 21/40 (52.5%) of clinically diagnosed sarcoids. More than half of 14 isolates (66.7%) shared 100% homology with BPV-1 Deltapapillomav...
Minkwitz C, Schoon HA, Zhang Q, Schöniger S.Equine endometrosis, a frequent cause of subfertility, is characterized by periglandular fibrosis, and no treatment exists. Endometrial biopsies not only contain diseased glands, but also contain healthy glands and stroma. Myoepithelial (ME) and myofibroblastic (MF) markers are calponin, smooth muscle actin (SMA), desmin and glial fibrillary acidic protein (GFAP). Epithelial vimentin expression indicates epithelial to mesenchymal transition (EMT). The aim of this immunohistochemical study was to investigate whether biopsies with endometrosis express MF and ME markers and vimentin. Compared to ...
Brasileiro LS, Segabinazzi LGTM, Menezes E, Salgueiro CC, Novello G, Scheeren VFDC, Alvarenga MA, Nunes JF.The aim of this study was to evaluate the effect of coconut water as a component of extender in different formulations for cooling equine sperm. One ejaculate of fourteen stallions was collected. Sperm was diluted to 50 × 10 sperm/mL using five different extenders: ACP-105: powdered coconut water extender (ACP-105, ACP Biotecnologia, Brazil); ACP-Milk: ACP-105 + 20 g/L of skimmed milk; ACP-EY 2.5%: ACP-105 + 2.5% of egg yolk; ACP-EY 5%: ACP-105 + 5% of egg yolk; and BotuSêmen (Botupharma, Botucatu, Brazil) and cooled in passive cooling device (BotuFlex, Botupharma, Botucatu, Brazil) at 5...
Hannan MA, Murase H, Sato F, Tsogtgerel M, Kawate N, Nambo Y.The peripheral blood concentrations of insulin-like peptide 3 (INSL3) have been detected in many mammalian species, but the level of INSL3 in horse remains unknown. The objectives were to develop a time-resolved fluorescence immunoassay (TRFIA) to detect INSL3 concentrations from horse blood as well as to determine the age-related and seasonal changes of plasma concentrations of INSL3 and testosterone from birth to early-puberty in Thoroughbred male horse (n = 11). Monthly blood sample and measurement of body weight, height, chest and cannon bone size were done from birth until 16 mo. Th...
Mattacola CG, Quintana C, Crots J, Tumlin KI, Bonin S. During thoroughbred races, jockeys are placed in potentially injurious situations, often with inadequate safety equipment. Jockeys frequently sustain head injuries; therefore, it is important that they wear appropriately certified helmets. The goals of this study are (1) to perform impact attenuation testing according to ASTM F1163-15 on a sample of equestrian helmets commonly used by jockeys in the United States and (2) to quantify headform acceleration and residual crush after repeat impacts at the same location. Seven helmet models underwent impact attenuation testing according to ASTM...
Legacki EL, Scholtz EL, Ball BA, Esteller-Vico A, Stanley SD, Conley AJ.Few studies have provided a longitudinal analysis of systemic concentrations of conjugated oestrogens (and androgens) throughout pregnancy in mares, and those only using immunoassay. The use of liquid chromatography tandem mass spectrometry (LC-MS/MS) will provide more accurate concentrations of circulating conjugated steroids. Objective: To characterise circulating concentrations of individual conjugated steroids throughout equine gestation by using LC-MS/MS. Methods: Longitudinal study and comparison of pregnant mares treated with vehicle or letrozole in late gestation. Methods: Sulphated oe...
Salem SE, Hough R, Probert C, Maddox TW, Antczak P, Ketley JM, Williams NJ, Stoneham SJ, Archer DC.Periparturient mares are at increased risk of colic including large colon volvulus, which has a high mortality rate. Alterations in colonic microbiota related to either physiological or management changes, or both, that occur at this time have been suggested as potential causes for increased colic risk in this population of horses. Although the effect of management changes on the horse faecal microbiota has been investigated, limited work has been conducted to investigate changes in faecal microbiota structure and function in the periparturient period. The objectives of the current study were ...
Mitchell CJ, O'Sullivan CM, Pinloche E, Wilkinson T, Morphew RM, McEwan NR.Next-generation sequencing of DNA from nematode eggs has been utilised to give the first account of the equine 'nemabiome'. In all equine faecal samples investigated, multiple species of Strongylidae were detected, ranging from 7.5 (SEM 0.79) with 99+% identity to sequences in the NCBI database to 13.3 (SEM 0.80) with 90+% identity. This range is typical of the number of species described previously in morphological studies using large quantities of digesta per animal. However, the current method is non-invasive; relies on DNA analysis, avoiding the need for specialist microscopy identificatio...
May JP, Walker CA, Maskell DJ, Slater JD.Streptococcus equi subsp. equi is the causative agent of the equine disease strangles. In this study we describe the development of an in vivo Himar1 transposon system for the random mutagenesis of S. equi and, potentially, other Gram-positive bacteria. We demonstrate efficient and random transposition of a modified mini-transposon onto the chromosome by Southern blot analysis and insertion site sequencing. Non-haemolytic mutants were isolated at a frequency of 0.2%, and acapsular mutants at a frequency of 0.04%. Taken together, these data demonstrate that in vivo Himar1 mutagenesis can be use...
Jacobsen S, Berg LC, Tvermose E, Laurberg MB, van Galen G.Neutrophil gelatinase-associated lipocalin (NGAL) has been shown to be a useful marker of kidney injury in people and dogs, but has not been described in horses. Objective: The aim of the study was to validate a commercially available porcine-specific ELISA to measure serum concentrations of equine NGAL. Methods: Intra- and interassay imprecisions were evaluated by multiple measurements on equine serum pools. Assay inaccuracy was determined by the linearity under dilution. Overlapping performance was assessed by measuring NGAL concentrations in horses with normal and elevated serum creatinine ...
Dell'Aquila ME, Caillaud M, Maritato F, Martoriati A, Gérard N, Aiudi G, Minoia P, Goudet G.The aim of this study was to investigate cumulus expansion, nuclear maturation and expression of connexin 43, cyclooxygenase-2 and FSH receptor transcripts in equine cumuli oophori during in vivo and in vitro maturation in the presence of equine FSH (eFSH) and precursors for hyaluronic acid synthesis. Equine cumulus-oocyte complexes (COC) were cultured in a control defined medium supplemented with eFSH (0 to 5 micrograms/ml), Fetal Calf Serum (FCS), precursors for hyaluronic acid synthesis or glutamine according to the experiments. After in vitro maturation, the cumulus expansion rate was incr...
Call GB, Wolfe MW.Activation of the luteinizing hormone beta (LHbeta) promoter by gonadotropin-releasing hormone (GnRH) via the transcription factor early growth response protein-1 (Egr1) has been well characterized. To determine the mechanisms affecting Egr1 regulation of LHbeta, we analyzed five different species of LHbeta promoters (equine, mouse, rat, bovine and human). Electrophoretic mobility shift assays (EMSAs) identified multiple transcription factors binding to the Egr regions on the LHbeta promoter. Species-specific differences existed in the binding affinity for Sp1, Sp3, steroidogenic factor-1 (SF-...
Irvine KL, Burt K, Hill AJ, Shaw S, Papasouliotis K.Equine pituitary pars intermedia dysfunction (PPID) may be diagnosed by measuring baseline plasma adrenocorticotrophic hormone (ACTH). The Immulite 1000 analyzer uses an automated chemiluminescence enzyme assay, previously validated for measuring equine ACTH. Recently, an automated bench-top immunoassay analyzer (AIA-360), designed for analytes in people, became available for veterinary use. Objective: Objectives were to evaluate analytic performance of the AIA immunoassay for measuring equine ACTH, and compare the results with those obtained by the Immulite. Methods: Adrenocorticotrophic horm...
Söderlund R, Bölske G, Holst BS, Aspán A.Infection by Mycoplasma felis is associated with ocular and respiratory disease in cats and respiratory disease in horses. A correct diagnosis is beneficial since the use of specific antimycoplasmal treatment can lead to resolution. The objective of the present study was to develop a real-time polymerase chain reaction (PCR) method based on dual-labeled fluorogenic probe technology, targeting the gene encoding elongation factor Tu (tuf ), for the fast and specific detection of M. felis. Specificity was achieved by basing the assay design on partial sequencing of the tuf gene in strains and cli...
Boschwitz JS, Timoney JF.The antiphagocytic property of equine fibrinogen for Streptococcus equi subsp. equi strain CF32 was examined in vitro. The results of bactericidal assays demonstrated that the presence of fibrinogen enhanced the ability of overnight and early log-phase cultures of strain CF32 to resist killing by equine neutrophils by 12-fold and seven-fold, respectively (p > 0.01). In addition, fibrinogen-coated bacteria treated with fibrinogen specific F(ab')2 fragments were 32% more susceptible to killing by equine neutrophils after opsonization in serum (p > 0.05), indicating that specific epitopes o...
Laroucau K, Colaneri C, Jaÿ M, Corde Y, Drapeau A, Durand B, Zientara S, Beck C.To evaluate the routine complement fixation test (CFT) used to detect Burkholderia mallei antibodies in equine sera, an interlaboratory proficiency test was held with 24 European laboratories, including 22 National Reference Laboratories for glanders. The panels sent to participants were composed of sera with or without B mallei antibodies. This study confirmed the reliability of CFT and highlighted its intralaboratory reproducibility. However, the sensitivity of glanders serodiagnosis and laboratory proficiency may be improved by standardising critical reagents, including antigens, and by dev...
Lancaster WD, Theilen GH, Olson C.DNAs from bovine papilloma virus(BPV)-induced hamster tumors and from equine connective tissue tumors of unknown etiology were examined for BPV DNA sequences by molecular hybridization. DNA from two distinct classes of BPV (type 1 and type 2) was labeled in vitro and used as probes. Analysis of DNA-DNA reassociation kinetics indicated that both virus types were capable of tumor induction in the hamster. DNA isolated from 6 of 7 equine tumors accelerated the reassociation of the BPV DNA probes. BPV type 1 or type 2 DNA hybridized extensively to DNA from 3 tumors, while 3 other tumors contained ...
Ricci G, Del Boccio G, Pennelli A, Aceto A, Whitehead EP, Federici G.Glutathione transferase (EC 2.5.1.18) from horse erythrocytes has been purified and some molecular and kinetic properties have been investigated. It appears to be a dimeric protein composed of subunits of about 23 kDa, indistinguishable either in sodium dodecyl sulfate or in urea electrophoresis. Amino acid composition, substrate specificities, sensitivity to inhibitors, CD spectra, and immunological studies provide evidence that the horse enzyme is related to the pi class transferases. This enzyme has only two reactive thiol groups/dimer whose integrity appears to be essential for the activit...
Morita H, Shiratori C, Murakami M, Takami H, Kato Y, Endo A, Nakajima F, Takagi M, Akita H, Okada S, Masaoka T.Two strains, KBL13(T) and GBL13, were isolated as one of intestinal lactobacilli from the faecal specimens from different thoroughbreds of the same farm where they were born in Hokkaido, Japan. They were Gram-positive, facultatively anaerobic, catalase-negative, non-spore-forming and non-motile rods. KBL13(T) and GBL13 homofermentatively metabolize glucose, and produce lactate as the sole final product from glucose. The 16S rRNA gene sequence, DNA-DNA hybridization, DNA G+C content and biochemical characterization indicated that these two strains, KBL13(T) and GBL13, belong to the same species...
Coleman M, Kuskie K, Liu M, Chaffin K, Libal M, Giguère S, Bernstein L, Cohen N.The objective of this study was to determine the in vitro antimicrobial activity of gallium maltolate (GaM) against Rhodococcus equi. A total of 98 virulent bacterial isolates from equine clinical cases were examined, of which 19 isolates were known to be resistant to macrolides and rifampin. Isolates were cultured with various concentrations of GaM and minimal inhibitory concentration (MIC) values were determined after 24 and 48 h. Both the MIC(50) and the MIC(90) after 24h of growth were 558 ng/mL (8 μM) and after 48 h of growth were 2230 ng/mL (32 μM). There were no apparent differences b...
Burrows R, Denyer M, Goodridge D, Hamilton F.Experimental ponies developed signs of disease four days after the intranasal instillation of A/England 1/79 equine influenza virus and virus was recovered from the nasopharynx from the second to the ninth day. No significant antigenic difference was found between the virus and the prototype A/Miami 1/63 virus, using post infection ferret and chicken sera and post vaccination pony sera. No antigenic differences were found between four viruses isolated between January and July 1979, although some differences were found in their ability to detect haemagglutination inhibiting antibody in convales...
McManus DP, Smyth JD.Extracts of the horse and sheep strains of Echinococcus granulosus and E. multilocularis were compared on the basis of their isoenzyme patterns for 10 enzymes by means of isoelectric focusing in polyacrylamide gels. The enzymes examined were: acid phosphatase, lactate dehydrogenase, malate dehydrogenase, malic enzyme, phosphoglucoseisomerase, isocitrate dehydrogenase, adenylate kinase, aldolase and alpha-glycerophosphate dehydrogenase. Interspecific and intraspecific differences are apparent in the isoenzyme profiles of all the enzymes except adenylate kinase; the pattern and activity of adeny...
Tözsér J, Friedman D, Weber IT, Bláha I, Oroszlan S.The proteinase of the equine infectious anemia virus (EIAV), a lentivirus closely related to human immunodeficiency virus (HIV), was purified from concentrated virus. The specificity of the enzyme was characterized using oligopeptides representing naturally occurring cleavage sites in the Gag and Gag-Pol polyproteins. The length of the substrate binding pocket was found to be 1-2 residues longer than that of HIV proteinases. Although the EIAV and HIV proteinases cleaved most of the peptides at the same bond, some were hydrolyzed by only the EIAV enzyme. Oligopeptides representing cleavage site...
Egito AS, Miclo L, López C, Adam A, Girardet JM, Gaillard JL.The equine alpha(s1)- and beta-caseins (CN) were purified by chromatography on DEAE-cellulose and by reversed-phase HPLC. The alpha(s1)-, beta-, and kappa-CN were characterized either by monodimensional urea-PAGE or sodium dodecylsulfate (SDS)-PAGE or by bidimensional electrophoresis. Kappa-casein was characterized after electrophoresis by glycoprotein-specific staining. To identify alpha(s1)-CN without ambiguity, internal sequences were determined after trypsin or chymosin digestion of purified alpha(s1)-CN. These sequences, that could be estimated to correspond to 62% of the full protein, pr...
Ascenzi P, di Masi A, Gullotta F, Mattu M, Ciaccio C, Coletta M.Cyanide binds to ferric heme-proteins with a very high affinity, reflecting the very low dissociation rate constant (k(off)). Since no techniques are available to estimate k(off), we report herewith a method to determine k(off) based on the irreversible reductive nitrosylation reaction to trap ferric myoglobin (Mb(III)). The k(off) value for cyanide dissociation from ferric cyanide horse heart myoglobin (Mb(III)-cyanide) was determined at pH 9.2 and 20.0 degrees C. Mixing Mb(III)-cyanide and NO solutions brings about absorption spectral changes reflecting the disappearance of Mb(III)-cyanide w...
Bundgaard L, Stensballe A, Elbæk KJ, Berg LC.Stem cells have great potential for tissue regeneration, but before stem cell populations can be used in the clinic, it is crucial that the stem cells have been definitely characterized by a set of specific markers. Although there have been attempts to identify a set of immunophenotypic markers to characterize equine mesenchymal stromal cells (MSCs), immunophenotyping of equine MSCs is still challenging due to the limited availability of suitable antibodies of high quality and consistent performance across different laboratories. The aim of this study was to evaluate a strategy for mapping the...
Tozaki T, Ohnuma A, Takasu M, Nakamura K, Kikuchi M, Ishige T, Kakoi H, Hirora KI, Tamura N, Kusano K, Nagata SI.Gene doping has raised concerns in human and equestrian sports and the horseracing industry. There are two possible types of gene doping in the sports and racing industry: (1) administration of a gene-doping substance to postnatal animals and (2) generation of genetically engineered animals by modifying eggs. In this study, we aimed to identify genetically engineered animals by whole-genome resequencing (WGR) for gene-doping control. Transgenic cell lines, in which the erythropoietin gene (EPO) cDNA form was inserted into the genome of horse fibroblasts, were constructed as a model of genetica...
Richard F, Messaoudi C, Lombard M, Dutrillaux B.Using human chromosome painting probes, we looked for homologies between human and mountain zebra (Equus zebra hartmannae, Equidae, Perissodactyla) karyotypes. Except for two very short segments, all euchromatic regions were found to have a human homologous chromosome segment. Conserved syntenies previously described in various mammalian orders were detected. Each synteny corresponded to a chromosomal region homologous to two parts of human chromosomes: HSA3 and HSA21, HSA7 and HSA16, HSA12 and HSA22, and HSA16 and HSA19. Chromosomal segments homologous to a part of HSA11 and HSA19p are found ...
Godard S, Schibler L, Oustry A, Cribiu EP, Guérin G.A horse BAC library was constructed with about 40,000 clones and mean insert size of 110 kb representing a 1.5 genome equivalent coverage and a probability of finding a single sequence of 0.75. It was characterized by PCR screening of about 130 sequences of horse microsatellites and exonic gene sequences retrieved from databases. BACs containing 8 microsatellites and 12 genes were subsequently localized by fluorescent in situ hybridization (FISH) on chromosomes. Two linkage groups were newly assigned to chromosomes: LG2 to ECA3 and LG5 to ECA24, and five linkage groups were also oriented--LG3,...
Gray WL, Yalamanchili R, Raengsakulrach B, Baumann RP, Staczek J, O'Callaghan DJ.Equine herpesvirus type 1 (EHV-1) preparations enriched in defective interfering particles (DIPs) have previously been demonstrated to mediate the coestablishment of persistent infection and oncogenic transformation in primary hamster embryo fibroblasts. In this study, it was demonstrated that infection of a rabbit kidney (RK) cell line with EHV-1 DIP-enriched preparations also results in the establishment of persistent infection. Viral transcription was characterized in RK cells infected with DIP-enriched stocks and compared to viral transcription in RK cells infected with standard (STD) EHV-...
Southgate CD, Green MR.Lentivirus Tat proteins comprise a novel class of RNA-binding transcriptional activators that are essential for viral replication. In this study, we performed a series of protein fusion experiments to delineate the minimal protein domains and promoter elements required for Tat action. We show that a 15-amino-acid region of equine infectious anemia virus (EIAV) Tat protein, when fused to the GAL4 or LexA DNA binding domain, can activate transcription in appropriate promoter contexts. In the natural human immunodeficiency virus type 1 long terminal repeat, activation by Tat is dependent on multi...
Williamson RM, Beveridge I, Gasser RB.To compare the sensitivities of three coprological techniques for the diagnosis of Anoplocephala perfoliata infection in horses and to assess the value of the methods for diagnosis of horses at risk of clinical cestodiasis. Methods: Faecal samples were collected from necropsied horses with or without A perfoliata infection and examined using one sedimentation and two different flotation methods. The coprological results were compared with worm counts performed at necropsy of the horses and the degree of mucosal damage. In addition, the efficiency of recovery of A perfoliata eggs from faeces wa...
Nash D, Lane E, Herath S, Sheldon IM.Endometritis after insemination is ubiquitous in the horse and is associated with semen and/or bacteria in the uterus. In up to 40% of horses, inflammation persists causing infertility. An endometrial explant culture was developed to study uterine secretion of prostaglandin F(2alpha) (PGF(2alpha)) in response to physiological and pathological challenge. Methods: Uteri were collected from mares, the endometrium dissected and explants from the uterine body or horn cultured in William's or RPMI medium. The response of explants to oxytocin, semen or bacteria compared to untreated tissue was tested...
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...
Tozaki T, Ohnuma A, Iwai S, Kikuchi M, Ishige T, Kakoi H, Hirota K, Kusano K, Nagata S.Gene doping is banned in human sports, horseracing, and equestrian sports. One possible form of gene doping is to administer exogenous genes, called transgenes. Several transgene detection methods based on quantitative PCR have been developed. In this study, we investigated the robustness of digital PCR and real-time PCR in transgene detection using primers and probes that matched (P-true) or incompletely matched (P-false) the template DNA. Fluorescence intensity was significantly reduced when substituted probes were used compared to that using the matched probe in both digital and real-time P...
Tang X, Sayre LM, Tochtrop GP.Cytochrome c is a key mitochondrial respiratory protein that is particularly susceptible to modification during oxidative stress. The nature of this susceptibility is linked to the mitochondrial membrane being rich in esterified linoleic acid, which predisposes this organelle to the formation of lipid peroxidation products such as 4-hydroxy-2-(E)-nonenal (4-HNE). To better understand the nature of cytochrome c modification by 4-HNE, we initiated an in vitro study utilizing a combination of MALDI-TOF mass spectrometry, LC-ESI-MS/MS and isotope labeling to monitor 4-HNE modification of cytochrom...
Malik P, Bálint A, Dán A, Pálfi V.Equine herpesvirus-1 (EHV-1) can be classified into distinct groups by single nucleotide polymorphisms (SNPs) in their genomes. Only a few of these can be associated with a special attribute of the virus. Differences in the ORF30 region can determine the neuropathogenic potential, while by substitutions in the ORF68 region several strain groups can be made. In previous studies no connection was found between the neuropathogenic potential and the SNPs in ORF68, but the occurrence of members of distinct groups in different outbreaks can facilitate epidemiological investigations because the geogr...
Yamagishi H, Nagamine T, Shimoda K, Ide S, Igarashi Y, Yoshioka I, Matumoto M.Single radial hemolysis (SRH), neutralization (NT), and hemagglutination inhibition (HI) tests were carried out on sera from horses immunized against the Prague and Miami strains of equine influenza virus. The HI and NT tests demonstrated good sensitivity; the sensitivity of the SRH test was somewhat lower. The NT titers of individual sera were correlated very closely with the HI titers, although the NT titers were higher. SRH zone diameters of individual sera also showed significant correlation with the NT and NI titers. The SRH test appears to be suitable for large-scale serological surveys ...
