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.
O'Rourke BA, Dennis JA, Healy PJ.Improvements to restriction fragment length polymorphism (RFLP)-based genotyping assays currently used for detection of mutations responsible for bovine ferrochelatase and myophosphorylase deficiencies, and equine hyperkalemic periodic paralysis (HYPP) are described. Reports of sporadic inhibition of restriction enzyme activity suggest a critical factor in RFLP-based genotyping assays should be assurance that restriction enzymes perform to specification with every sample. The RFLP genotyping assays that use either a mismatched recognition sequence in one or both of the oligonucleotides, or inc...
Huang X, Xuan X, Yokoyama N, Katayama Y, Anzai T, Igarashi I.Two enzyme-linked immunosorbent assays (ELISA) with recombinant protein as antigens were evaluated by comparison with the indirect fluorescent antibody tests (IFAT) for the detection of specific antibodies to Babesia caballi and Babesia equi, respectively in 380 sera from experimentally infected, uninfected, and field horses. The high concordances of 92.4% (351/380) and 98.2% (373/380) between ELISA and IFAT for B. caballi and B. equi, respectively suggest that ELISA, especially for B. equi infection, could be alternative to the corresponding IFAT for serodiagnoses of equine piroplasmosis, alt...
Leeb T, Vogl C, Zhu B, de Jong PJ, Binns MM, Chowdhary BP, Scharfe M, Jarek M, Nordsiek G, Schrader F, Blöcker H.In an effort to increase the density of sequence-based markers for the horse genome we generated 9473 BAC end sequences (BESs) from the CHORI-241 BAC library with an average read length of 677 bp. BLASTN searches with the BESs revealed 4036 meaningful hits (E <or= 10(-5)) in the human genome that provide useful markers for the human-horse comparative map. The 4036 BLASTN hits allowed the anchoring of 3079 BAC clones to the human genome, on average one corresponding equine BAC clone per megabase of human DNA. We used the BLASTN anchored BESs for an in silico prediction of the gene content an...
Maes A, Baert K, Croubels S, De Clercq D, van Loon G, Deprez P, De Backer P.A rapid method for the quantification of amiodarone and desethylamiodarone in animal plasma using high-performance liquid chromatography combined with UV detection (HPLC-UV) is presented. The sample preparation includes a simple deproteinisation step with acetonitrile. In addition, a sensitive method for the quantification of amiodarone and desethylamiodarone in horse plasma and urine using high-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is described. The sample preparation includes a solid-phase extraction (SPE) with a SCX c...
Leclere M, Desnoyers M, Beauchamp G, Lavoie JP.Mast cells normally are present in equine bronchoalveolar lavage fluid (BALF), but usually represent <2% of all cells in healthy horses. An increased percentage of mast cells has been associated with airway hyperactivity and inflammatory airway diseases, but marked differences are reported between studies in normal and diseased horses. Because an abnormal mast cell count may be of clinical relevance, we compared the ability of a fast Romanowsky method to stain mast cell granules with that of 3 metachromatic stains: automated Romanowsky, May-Grünwald Giemsa, and toluidine blue stains. The B...
White SD, Vandenabeele SI, Drazenovich NL, Foley JE.Malassezia-type yeasts previously have been observed on cytologic examination of the intermammary region of mares that presented with tail-head pruritus; topical antiyeast treatment resolved the pruritus. Further, Malassezia dermatitis has been observed in horses in intertriginous areas such as the udder and prepuce; the species of yeast was not confirmed. It is not known whether healthy mares or male horses can be carriers of this yeast in these body areas. Objective: Malassezia spp. are present in the intermammary region in healthy mares and the preputial fossa in healthy geldings. Methods: ...
Breuhaus BA, Refsal KR, Beyerlein SL.The purpose of the study reported here was to validate measurement of free thyroxine (fT4) concentration in equine serum by equilibrium dialysis (fT4D), and to compare values with fT4 concentration measured directly and with total T4 (TT4) concentration. The fT4D, fT4, and TT4 concentrations were measured over a range of values in euthyroid horses and horses made hypothyroid by administration of propylthiouracil (PTU). Concentrations of fT4D (<1.8-83 pmol/L) were consistently higher than those of fT4 (<1-40 pmol/L). There was a significant (P < .001) regression of fT4D on fT4 in 503 s...
Smith JJ, Ross MW, Smith RK.The purpose of this study was to investigate the response of suspensory ligament fibroblasts (SLF) to in vitro stimulation using acellular bone marrow (ABM), platelet rich plasma (in vitro PRP), and serum as potential treatment modalities for suspensory desmitis. Blood, bone marrow, and suspensory ligaments were collected from five horses. SLF were harvested, grown until confluent, and stimulated with various concentrations of ABM, PRP, equine serum, foetal bovine serum, and medium (control). The responses to the treatments were assessed using a combination of radio-labeling for total protein ...
Morresey PR, Mackay RJ.To measure serum polymyxin B concentration after single and repeated IV infusions in horses. Methods: 5 healthy horses. Methods: In study 1, 1 mg (6,000 U) of polymyxin B/kg was given IV and blood samples were collected for 24 hours. In study 2, 1 mg of polymyxin B/kg was given IV every 8 hours for 5 treatments and blood samples were collected until 24 hours after the last dose. Polymyxin B concentration was measured as the ability to suppress nitrite production by murine macrophages stimulated with lipopolysaccharide and interferon-alpha. Urine was collected prior to the first drug infusion a...
Ainsworth DM, Wagner B, Franchini M, Grünig G, Erb HN, Tan JY.To evaluate time-dependent alterations in gene expression of chemokines in bronchial epithelium of recurrent airway obstruction (RAO)-affected horses and whether alterations resulted from increases in gene expression of interleukin (IL)-17 in cells isolated from bronchoalveolar lavage fluid (BALF). Methods: 8 RAO-susceptible horses and 9 control horses. Methods: In 2 experiments, both groups of horses were evaluated after being maintained on pasture and after being stabled and fed dusty hay for 1, 14, 35, and 49 days (experiment 1) or 14 and 28 days (experiment 2). In experiment 1, gene expres...
Weinstein WL, Moore PA, Sanchez S, Dietrich UM, Wooley RE, Ritchie BW.To determine whether a novel third-generation chelating agent (8 mM disodium EDTA dehydrate and 20 mM 2-amino-2-hydroxymethyl-1, 3-propanediol) would act as an antimicrobial potentiator to enhance in vitro activity of antifungal medications against fungal isolates obtained from horses with mycotic keratitis. Methods: Fungal isolates (3 Aspergillus isolates, 5 Fusarium isolates, 1 Penicillium isolate, 1 Cladosporium isolate, and 1 Curvularia isolate) obtained from horses with mycotic keratitis and 2 quality-control strains obtained from the American Type Culture Collection (ATCC; Candida albica...
Sabeur K, Ball BA.To identify the generation of the superoxide anion by equine spermatozoa. Methods: Multiple ejaculates collected from 3 Thoroughbred stallions. Methods: Induced superoxide production by reduced nicotinamide adenine dinucleotides (NAD[P]H; ie, reduced nicotinamide adenine dinucleotide [NADH] and reduced nicotinamide adenine dinucleotide phosphate [NADPH]) was measured by use of a nitroblue tetrazolium (NBT) reduction assay on whole spermatozoa and a cytochrome c reduction assay on isolated membrane fractions of spermatozoa. Localization of superoxide generation was determined by use of NBT cyto...
Perrocheau M, Boutreux V, Chadi S, Mata X, Decaunes P, Raudsepp T, Durkin K, Incarnato D, Iannuzzi L, Lear TL, Hirota K, Hasegawa T, Zhu B, de Jong P....A medium-density map of the horse genome (Equus caballus) was constructed using genes evenly distributed over the human genome. Three hundred and twenty-three exonic primer pairs were used to screen the INRA and the CHORI-241 equine BAC libraries by polymerase chain reaction and by filter hybridization respectively. Two hundred and thirty-seven BACs containing equine gene orthologues, confirmed by sequencing, were isolated. The BACs were localized to horse chromosomes by fluorescent in situ hybridization (FISH). Overall, 165 genes were assigned to the equine genomic map by radiation hybrid (RH...
Shahar R, Zaslansky P, Barak M, Friesem AA, Currey JD, Weiner S.Young's modulus and Poisson's ratios of 6mm-sized cubes of equine cortical bone were measured in compression using a micro-mechanical loading device. Surface displacements were determined by electronic speckle pattern-correlation interferometry. This method allows for non-destructive testing of very small samples in water. Analyses of standard materials showed that the method is accurate and precise for determining both Young's modulus and Poisson's ratio. Material properties were determined concurrently in three orthogonal anatomic directions (axial, radial and transverse). Young's modulus va...
Lopez I, Estepa JC, Mendoza FJ, Mayer-Valor R, Aguilera-Tejero E.To establish reference values for protein-bound, ionized, and weak-acid complexed fractions of calcium and magnesium in equine serum and determine stability of ionized calcium (iCa) and ionized magnesium (iMg) in serum samples kept under various storage conditions. Methods: 28 clinically normal horses. Methods: Total calcium (tCa) and magnesium (tMg) in equine serum were fractionated by use of a micropartition system that allows separation of protein-bound calcium (pCa) and magnesium (pMg) and ultrafiltrable calcium (microCa) and magnesium (microMg) fractions. Serum concentrations of iCa and i...
Pusterla N, Madigan JE, Leutenegger CM.The focus of rapid diagnosis of infectious disease of horses in the last decade has shifted from the conventional laboratory techniques of antigen detection, microscopy, and culture to molecular diagnosis of infectious agents. Equine practitioners must be able to interpret the use, limitations, and results of molecular diagnostic techniques, as they are increasingly integrated into routine microbiology laboratory protocols. Polymerase chain reaction (PCR) is the best-known and most successfully implemented diagnostic molecular technology to date. It can detect slow-growing, difficult-to-cultiv...
Wagner ML, Raudsepp T, Goh G, Agarwala R, Schaffer AA, Dranchak PK, Brinkmeyer-Langford C, Skow LC, Chowdhary BP, Mickelson JR.A comparative approach that utilizes information from more densely mapped or sequenced genomes is a proven and efficient means to increase our knowledge of the structure of the horse genome. Human chromosome 2 (HSA2), the second largest human chromosome, comprising 243 Mb, and containing 1246 known genes, corresponds to all or parts of three equine chromosomes. This report describes the assignment of 140 new markers (78 genes and 62 microsatellites) to the equine radiation hybrid (RH) map, and the anchoring of 24 of these markers to horse chromosomes by FISH. The updated equine RH maps for ECA...
Gall D, Nielsen K, Bermudez RM, Muñoz del Real MC, Halbert G, Groulx R, Moreno F, Chow EY, Checkley SL.An indirect enzyme-linked immunosorbent assay (IELISA) was developed for the detection of equine serum antibodies to lipopolysaccharide of Salmonella enterica subsp. enterica serovar Abortusequi (LPS), a causative organism of Equine Paratyphoid. The data presented demonstrates that horses immunized with S. abortusequi LPS developed antibodies detectable by the IELISA. By comparison, the tube agglutination test (TAT) did not detect antibody to S. abortusequi LPS as consistently as the IELISA. The data suggests that the IELISA may be a more suitable test for the detection of serum antibodies to ...
Vilasi S, Dosi R, Iannuzzi C, Malmo C, Parente A, Irace G, Sirangelo I.In protein deposition disorders, a normally soluble protein is deposited as insoluble aggregates, referred to as amyloid. The intrinsic effects of specific mutations on the rates of protein aggregation and amyloid formation of unfolded polypeptide chains can be correlated with changes in hydrophobicity, propensity to convert alpha-helical to beta sheet conformation and charge. In this paper, we report the aggregation rates of buffalo, horse and bovine apomyoglobins. The experimental values were compared with the theoretical ones evaluated considering the amino acid differences among the sequen...
Werners AH, Bull S, Vendrig JC, Smyth T, Bosch RR, Fink-Gremmels J, Bryant CE.The inter- and intra-species differences in the response to lipopolysaccharides (LPS) are well recognised in mammalian species. It has been hypothesized that these differences can be attributed to genetic polymorphisms in the components involved in LPS signal transduction. These components include the cluster of differentiation factor 14 (CD-14), a membrane bound protein on the surface of mononuclear cells that recognises LPS and a receptor complex consisting of Toll-like receptor-4 (TLR-4) and myeloid differentiation factor-2 (MD-2). Sequencing of these three proteins in humans and mice revea...
Wagner B, Hillegas JM, Antczak DF.Interleukin-4 (IL-4) is secreted by T helper type 2 cells, mast cells, basophils and eosinophils. Detection of IL-4 can contribute the evaluation of cellular immune responses during infectious diseases, immunological disorders or vaccination. We used recombinant equine IL-4 to generate a monoclonal antibody (mAb) to equine IL-4. The mAb detected recombinant IL-4 in mammalian cells transfected with different plasmids containing IL-4 cDNA. After mitogen stimulation of equine peripheral blood mononuclear cells, an intracellular protein was recognized by the new mAb in 1-2% of lymphocytes using fl...
Scholz HC, Joseph M, Tomaso H, Al Dahouk S, Witte A, Kinne J, Hagen RM, Wernery R, Wernery U, Neubauer H.A polymerase chain reaction (PCR) assay targeting the flagellin P (fliP)-I S407A genomic region of Burkholderia mallei was developed for the specific detection of this organism in pure cultures and clinical samples from a recent outbreak of equine glanders. Primers deduced from the known fliP-IS407A sequence of B. mallei American Type Culture Collection (ATCC) 23344(T) allowed the specific amplification of a 989-bp fragment from each of the 20 B. mallei strains investigated, whereas other closely related organisms tested negative. The detection limit of the assay was 10 fg for purified DNA of ...
Tanner JM, Traub-Dargatz JL, Hill AE, Van Campen H, Knight AP, Cunningham WE, Salman MD.To describe the prevalence of West Nile virus (WNV) infection and evaluate factors associated with positive IgM capture ELISA results in equids with clinical signs compatible with WNV infection. Methods: Retrospective case series. Methods: Laboratory submission forms from 1,104 equids tested for WNV in Colorado in 2003. Methods: Submission forms accompanying samples submitted for detection of WNV via IgM capture ELISA were obtained from the Colorado state veterinarian and diagnostic laboratories performing the tests. Data on signalment, clinical signs, history of vaccination against WNV, and a...
Tepliakova TV, Efremova EA, Riabchikova EI.The carnivorous fungi hyphomycetes are natural enemies of soil nematodes. Laboratory tests examining the effect of the effective strain Duddingtonia flagrans T-89 on equine strongyle larvae have indicated that their size can be reduced 5-48-fold under the action of the fungus. Using helminth-infected mice as an example has ascertained that when the animals are fed a biopreparation, the chlamydial spores of the carnivorous fungus D. flagrans remain viable and continue their development in the excrements. The dead nematodes show cell structural impairments in all tissues and organs, which may be...
Dahlgren LA, Mohammed HO, Nixon AJ.he treatment of overuse tendon injuries with exogenous growth factors such as insulin-like growth factor-I (IGF-I) may facilitate an improved return to sustained athletic function. The biological effects of IGF-I are exerted under the control of a complex of IGF receptors, binding proteins, and proteases. This IGF system includes a family of six structurally related high-affinity IGF binding proteins (IGFBPs) that protect IGF-I from local proteases and restrict access of IGF-I to its receptor. This study describes the expression of the IGFBPs in flexor tendon after acute injury and during heal...
Schejter A, Ryan MD, Blizzard ER, Zhang C, Margoliash E, Feinberg BA.Contrary to most heme proteins, ferrous cytochrome c does not bind ligands such as cyanide and CO. In order to quantify this observation, the redox potential of the ferric/ferrous cytochrome c-cyanide redox couple was determined for the first time by cyclic voltammetry. Its E0' was -240 mV versus SHE, equivalent to -23.2 kJ/mol. The entropy of reaction for the reduction of the cyanide complex was also determined. From a thermodynamic cycle that included this new value for the cyt c cyanide complex E0', the binding constant of cyanide to the reduced protein was estimated to be 4.7 x 10(-3) L M(...
Weston MC, Collins ME, Cunningham FM.To assess the biological effects of purified recombinant equine CCL11 on equine eosinophil function. Methods: Following stimulation of eosinophils from normal horses, the polymerised form of actin was measured by flow cytometry using fluorescently labelled phalloidin. Migration was determined in a 96 well plate chemotaxis assay using 8 microm pore membranes, and adherence of eosinophils to serum-coated plastic was assessed using a colorimetric assay for eosinophil peroxidase. Superoxide generation was measured by the reduction of cytochrome C in a colorimetric assay. Results: Equine CCL11 indu...
Hodgkinson JE.The future implementation of improved and sustainable control strategies for the major equine parasites will be dependent on a greater insight into their basic biology, pathogenicity and epidemiology together with an enhanced ability for accurate diagnosis. This paper will provide a review of the current molecular methods under development for the detection of equine parasites and their application to current scientific questions. In particular, the strongyles are recognised as important pathogens of horses and recent advances made in the study of this parasitic group at the single species lev...
Mills PC, Cross SE.Little is known about the transdermal penetration of hydrocortisone in the horse and, although commercial formulations containing hydrocortisone are registered for topical use in the horse, there have been no studies investigating the movement of this glucocorticoid through different regions of equine skin. Skin was harvested from the thorax, groin and leg (dorsal metacarpal) regions of five Thoroughbred geldings and frozen (-20 degrees C) until required. Defrosted skin was placed in Franz-type diffusion cells and the amount of radiolabelled ((3)H) hydrocortisone, in a saturated solution of un...
Coles GC, Jackson F, Pomroy WE, Prichard RK, von Samson-Himmelstjerna G, Silvestre A, Taylor MA, Vercruysse J.Before revised World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines on the detection of anthelmintic resistance can be produced, validation of modified and new methods is required in laboratories in different parts of the world. There is a great need for improved methods of detection of anthelmintic resistance particularly for the detection of macrocyclic lactone resistance and for the detection of resistant nematodes in cattle. Therefore, revised and new methods are provided here for the detection of anthelmintic resistance in nematodes of ruminants, horses and ...
Gutmann S, Zawatzky R, Müller M.Interferon-gamma (IFN-gamma) is a key cytokine in cell-mediated immunity. To measure IFN-gamma production of equine lymphocytes (eqIFN-gamma), we developed a quantitative ELISA. Monoclonal antibodies (mAb) were produced against bacterially derived eqIFN-gamma. The mAbs recognised recombinant and lymphocyte-derived eqIFN-gamma in ELISA, Western blotting, as well as flow cytometric and microscopic analysis. In contrast to bacterially derived material, mammalian and insect cell-derived eqIFN-gamma was biologically active but could be neutralised by one of the monoclonal antibodies. Unexpectedly, ...
Posnett ES, Ambrosio RE.This report describes DNA probes for the identification of Babesia equi. A genomic library of B. equi was constructed in pUC13. Several clones were identified that hybridized strongly to B. equi DNA. Clone pBE33 hybridized specifically to B. equi DNA and did not hybridize to horse DNA nor to DNA from Babesia caballi, Babesia bovis or Babesia bigemina. Two subclones of pBE33 (pSB20 and pEH21) containing B. equi repetitive sequences, could detect 0.49 ng and 0.97 ng B. equi DNA, respectively.
Pan W, Shen Z, Wang H, He H.Vesicular stomatitis virus (VSV) is an archetypal member of Mononegavirales which causes important diseases in cattle, horses and pigs. The matrix protein (M) of VSV plays critical roles in the replication, assembly/budding and pathogenesis of VSV. To further investigate the role of M during viral growth, we used a two-hybrid system to screen for host factors that interact with the M protein. Here, NADH: ubiquinone oxidoreductase complex assembly factor 4 (Ndufaf4) was identified as an M-binding partner, and this interaction was confirmed by yeast cotransformation and GST pulldown assays. ...
Barteri M, Gaudiano MC, Santucci R.The influence of glycerol on the structural properties of Fe(III)-horse heart myoglobin has been investigated by absorbance, CD and SR-SAXS spectroscopy. The results obtained indicate that both the tertiary and the secondary (alpha-helix) conformations of the protein are influenced by glycerol; in particular, an increase of approx. 8% in helical content was observed. Further, analysis of both the acid- and guanidine-induced denaturation transitions points to a glycerol-induced decreased stability of the tertiary structure; conversely, the alpha-helix conformation is found to be stabilized by t...
Puppione DL, Whitelegge JP, Yam LM, Bassilian S, Schumaker VN, MacDonald MH.In pigs, humans, chimpanzees and probably other great apes, a cysteine at residue 6 enables apolipoprotein A-II to form a homodimer. However, the apoA-IIs of other primates, lacking a cysteine residue, are monomeric. We have already reported that horse apoA-IIs form homodimers due also to a cysteine at residue 6. In this study, we wanted to determine whether other equine apoA-IIs might be monomeric. The high density lipoproteins were ultracentrifugally isolated from the plasmas of a horse (Equus caballus), a donkey (Equus asinus) and five wild equines: two types of zebras (Equus zebra hartmann...
Rossi TM, Pyle WG, Maxie MG, Pearl DL, Physick-Sheard PW.In 2000, troponin assays were adopted as the test of choice for detection of myocardial injury in man. This decision was made after extensive testing and followed a 60 year search for a biomarker of myocardial damage with sufficient analytical sensitivity and specificity. This has led to proliferation of assays for use in human medicine, each requiring extensive testing and validation before it could be made available on the open market for human use. The search for ever-more analytically sensitive assays and for a standard reference material continues. The adoption of troponin testing in vete...
Ma Y, Ke C, Wan Z, Li Z, Cheng X, Wang X, Zhao J, Ma Y, Ren L, Han H, Zhao Y.The neonatal Fc receptor (FcRn) is involved in IgG metabolism and transport in placental mammals. However, whether FcRn is responsible for IgG transfer from maternal serum to colostrum/milk is controversial. Interestingly, large domestic animals, such as cows, pigs, sheep, and horses, in which passive IgG transfer is exclusively completed via colostrum/milk, all express an FcRn α-chain that is shorter in the cytoplasmic tail (CYT) than its counterparts in humans and rodents. To address whether the length variation has any functional significance, we performed in vitro experiments using the Tr...
De Keyser K, Berth M, Christensen N, Willaert S, Janssens S, Ducatelle R, Goddeeris BM, De Cock HE, Buys N.Diagnosis of chronic progressive lymphoedema (CPL) in draught horses, including the Belgian Draught Horse, is mainly based on clinical evaluation of typical lower limb lesions. A deficient perilymphatic elastic support, caused by a pathological elastin degradation in skin and subcutis, has been suggested as a contributing factor for CPL. Elastin degradation products induce the generation of anti-elastin Ab (AEAb), detectable in horse serum by ELISA. For a clinically healthy group of draught horses, a significantly lower average AEAb-level than 3 clinically affected groups (mild, moderate and s...
Klingeborn B, Dinter Z.Antibody to equid herpesvirus 1, which mediates single radial hemolysis, is that responsible for neutralization. Hemagglutination inhibition antibody is not necessarily involved in neutralization or hemolysis.
Varner DD, Ward CR, Storey BT, Kenney RM.Equine spermatozoa were incubated in a chemically defined medium for 8 hours. The medium preserved spermatozoal viability, as assessed by total spermatozoal motility, progressive spermatozoal motility, and spermatozoal exclusion of eosin stain. Effects of time and divalent cation ionophore, A23187, on the occurrence and character of the spermatozoal acrosome reaction were determined. Two light microscopic assays, a triple-stain technique and a chlortetracycline fluorescence assay, were calibrated with transmission electron microscopy for detection of the acrosome reaction. Incubation time and ...
Kútvölgyi G, Stefler J, Kovács A.A simple trypan blue-neutral red-Giemsa staining procedure for simultaneous evaluation of acrosome, sperm head, and tail membrane integrity and morphology has been used to evaluate equine spermatozoa. Some special characteristics and problems have arisen in evaluating stallion semen. One problem was the differentiation of intact vs. damaged sperm tails primarily in frozen and thawed samples. After freezing and thawing, a high percentage of spermatozoa with an unstained head and stained tail were observed. These cells are considered immotile. Therefore, unambiguous differentiation of intact vs....
Sankaranarayanan R, Biswal BK, Vijayan M.A new relaxed state has been characterized in the crystals of horse methemoglobin grown at neutral pH at low ionic concentration and their low humidity variants. The crystals provide an example for improvement in X-ray diffraction quality with reduced solvent content. Only the classical R state has been so far observed in liganded horse hemoglobin. The state characterized in the present study lies in between the R state and the R2 state characterized earlier in liganded human hemoglobin. The results presented here, along with those of earlier studies, suggest that relaxed and tense hemoglobin ...
Sternberg S.Swab samples were collected from the oral cavity of 174 horses in 10 farms and cultured selectively for Actinobacillus equuli. A. equuli could be isolated from 37% of all samples, varying between 12 and 88% in the different farms. Eight horses were sampled repeatedly for several days, with a variation in isolation frequency between 50 and 88%. Isolates were compared by restriction enzyme digestion and Pulsed-Field Gel Electrophoresis. A high degree of strain variability was found within each horse population as well as some variability over time between strains isolated from the same horse.
Fujita K, Suzuki I, Ochiai J, Shinpo K, Inoue S, Saito H.We found that aloe extract contains a lectin-like substance which reacts with serum proteins of various animals. Furthermore, in human serum 2 proteins, alpha2-macroglobulin and alpha1-antitrypsin, were shown to be reactive with aloe extract.
Palacios H, Hibarren I, Olalla MJ, Cala V.The diagnosis of lead poisoning in horses living on farmland in the vicinity of a battery recycling plant was based on clinical signs as well as on laboratory findings. Chemical analysis of six surface soils (0-15 cm) and herbage samples taken at different distances to the recycling plant showed very high total lead levels in the closest sites to the facility and a clear decrease with distance. Total lead levels in soil samples ranged from 127 to 5657 mg kg(-1), with more than 70% of lead extractable by EDTA in the most polluted soils. Lead levels in the aerial part of herbage samples were in ...
Wang T, Magnarelli LA, Anderson JF, Gould LH, Bushmich SL, Wong SJ, Fikrig E.Recombinant West Nile virus envelope (E) protein was examined in enzyme-linked immunosorbent assay (ELISA) to detect antibodies elicited during West Nile virus infection. Horses (nine of 10) and humans (six of six) with confirmed West Nile virus infection had IgG and/or IgM antibodies to the E protein. Antibodies to the recombinant West Nile virus membrane and nonstructural 1 proteins were not detected in any of these sera. An E protein-based ELISA may aid in the serological diagnosis of West Nile virus infection.
Main AR, McKnelly SC, Burgess-Miller SK.A butyrylcholinesterase of mol.wt. approx. 83000 was observed in pooled rabbit serum. The enzyme was named monomeric butyrylcholinesterase to distinguish it from the larger oligomeric butyrylcholinesterase of horse and human serum whose subunits are the same size as the monomeric enzyme. The active-site concentration of monomeric butyrylcholinesterase in the pooled serum was 0.18mum, which is five times the concentration of butyrylcholinesterase in pooled horse serum. This was surprising, since the horse serum is regarded as a rich source of butyrylcholinesterase, whereas rabbit serum is not g...
Rader K, Choi YH, Hinrichs K.Intracytoplasmic sperm injection is becoming a common clinical procedure in the horse, but little information is available on techniques for its performance. Each laboratory uses different procedures and different media for the steps involved with in vitro embryo production. This article outlines the procedures used in the Clinical Equine Intracytoplasmic Sperm Injection Program at Texas A&M University for in vitro blastocyst production during the past 3 years.
Meneses CS, Müller HY, Herzberg DE, Uberti B, Bustamante HA, Werner MP.The role of glial cells in pain modulation has recently gathered attention. The objective of this study was to determine healthy spinal microglia and astrocyte morphology and disposition in equine spinal cord dorsal horns using Iba-1 and GFAP/Cx-43 immunofluorescence labeling, respectively. Five adult horses without visible wounds or gait alterations were selected. Spinal cord segments were obtained post-mortem for immunohistochemical and immunocolocalization assays. Immunodetection of spinal cord dorsal horn astrocytes was done using a polyclonal goat antibody raised against Glial Fibrillary ...
Dumasia MC, Houghton E, Bradley CV, Williams DH.The metabolism and urinary excretion of 1,2(n)-3H-1-dehydrotestosterone were studied in cross-bred gelded horses. Approximately 40% of the dose was excreted in 24 h. The steroid metabolites were extracted by Amberlite XAD-2 resin and fractionated into glucuronides and sulphoconjugates. Unchanged 1-dehydrotestosterone was the only component identified by gas chromatography mass spectrometry after solvolysis of the sulphoconjugates. Positive and negative ion fast atom bombardment mass spectra were obtained on the purified 1-dehydrotestosterone sulphoconjugate isolated from horse urine and on the...
Wickhorst JP, Sammra O, Hassan AA, Alssashen M, Lämmler C, Prenger-Berninghoff E, Erhard M, Metzner M, Paschertz K, Timke M, Abdulmawjood A.In the present study an Arcanobacterium hippocoleae strain isolated from a uterus swab of an apparently healthy mare could be identified by phenotypic properties, by MALDI-TOF MS analysis and genotypically by investigating the molecular targets 16S rDNA, 16S-23S rDNA intergenic spacer region and the genes encoding the β subunit of bacterial RNA polymerase (rpoB), elongation factor tu (tuf) and glyceraldehyde 3-phosphate dehydrogenase (gap). The presented data are one of the few reports about the species A. hippocoleae and might help to elucidate the role this species plays in infections of ho...
Moore T, Wilcke J, Chilcoat C, Eyre P, Crisman M.Equine neutrophils (PMN) play a critical role in inflammatory processes in horses. The objective of this study was to characterize equine PMN function ex vivo following stimulation with calcium ionophore A23187 (A23187) and phorbol myristate acetate (PMA). These stimulants trigger different branches of the PMN activation process that occurs in vivo. Equine PMN were isolated from the whole blood of six clinically normal geldings using a one-step discontinuous Percoll gradient technique. Neutrophil aggregation, degranulation, and superoxide anion production were evaluated in assay systems which ...
Edlund O, Bowers L, Henion J, Covey TR.Urine samples were spiked with [17-methyl-2H3]methandrostenolone as internal standard and extracted with a mixture of dichloromethane and cyclohexane. The organic phase was concentrated and injected onto a short octyl-silica column (30 mm x 4.6 mm I.D.) for separation of methandrostenolone and 17-epimethandrostenolone. The effluent from the column was connected to a Sciex TAGA 6000E triple quadrupole mass spectrometer equipped with an atmospheric pressure ion source for sampling of ions generated by a heated pneumatic nebulizer with corona discharge ionization. This ion source produced abundan...
Jinglong X, Xiaobin L, Fang Z, Chenchen W, Kailun Y.The aim of this research was to isolate bacteria capable of biotransforming daidzein from fresh feces from pregnant horses. A Hungate anaerobic roller tube was used for anaerobic culture. Single colonies were picked at random and incubated with daidzein. High performance liquid chromatography was used to detect whether the isolated bacteria were able to biotransform the substrate. A strain capable of reducing daidzein was selected and characterized using sequence analysis of 16S rDNA, and a phylogenetic tree was constructed. The morphological physiological and biochemical characteristics of th...
Okano S, Hurley DJ, Vandenplas ML, Moore JN.To determine the effect of fetal bovine serum (FBS) and heat-inactivated FBS (HI-FBS) on lipopolysaccharide (LPS)- and zymosan-induced procoagulant activity of equine and canine mononuclear cells. Methods: Mononuclear cells from 18 horses and 3 dogs. Methods: Cells were incubated with various concentrations of FBS, HI-FBS, LPS, zymosan, polymyxin B, and anti-LPS-binding protein monoclonal antibody or combinations of these constituents. A 1 stage recalcification assay was used to determine procoagulant activity. Results: Addition of FBS to media significantly increased procoagulant activity; eq...
Rubio C, Cubillo MA, Hooghuis H, Sanchez-Vizcaino JM, Diaz-Laviada M, Plateau E, Zientara S, Crucière C, Hamblin C.The mortality rate in susceptible populations of horses during an epizootic of African horse sickness (AHS) may be in excess of 90%. Rapid and reliable assays are therefore essential for the confirmation of clinical diagnoses and to enable control strategies to be implemented without undue delay. One of the major objectives of a recent European Union funded project was the validation of newly developed diagnostic assays which are rapid, sensitive, highly reproducible and inexpensive, for the detection of African horse sickness virus (AHSV) antigens and antibodies. The Laboratorio de Sanidad y ...
Relave F, Lefebvre RC, Beaudoin S, Price C.The aim of this study was to validate an enzyme-linked immunosorbent assay (ELISA) for the measurement of progesterone (P4) in mares. Specifically, the objectives were as follows: (1) to determine the specificity and sensitivity of the ELISA test for determination of P4, (2) to measure the potential agreement between the 2 people performing the test, and 3) to evaluate the effect of time on the outcome. Ten mares were sampled on the day before ovulation (D-1), and on days 1 (D1), 3 (D3), and 5 (D5) following ovulation, during the reproductive season. While mares were cycling regularly, estrus ...
Klymiuk MC, Speer J, Marco I, Elashry MI, Heimann M, Wenisch S, Arnhold S.Osteoarthritis (OA) is a common and incurable disease in humans and animals. To gain a better understanding of the pathogenesis and identify potential treatments, miRNAs will be extracted and analysed from extracellular vesicles (EVs) of equine adipose derived mesenchymal stem cells (AdMSCs). Methods: For this purpose we cultivated and pretreated AdMSCs under different conditions: interleukin 1β, shock wave, chondrogenic differentiation, chondrogenic differentiation under hypoxia, or after senescence. After treatment, EVs were harvested from the cell culture supernatants. Next-generation sequ...
Maeda K, Kai K, Matsumura T.Infection with equine herpesvirus-4 (EHV-4) is a major cause of respiratory tract disease, equine rhinopneumonitis, in horses. Although the full sequence of EHV-4 has been reported, genomic differences among EHV-4 field isolates have not yet been characterized. In this study, the genomic diversity between 23 Japanese EHV-4 isolates was analyzed by digestion with restriction endonucleases (BamHI, BgIII, EcoRI, SacI, and SalI) and polymerase chain reaction (PCR). The restriction endonuclease digestion patterns of the EHV-4 field isolates showed distinct differences which included mobility shifts...
