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.
González-Fernández L, Macías-García B, Loux SC, Varner DD, Hinrichs K.Protein tyrosine phosphorylation (PY) is a hallmark of sperm capacitation. In stallion sperm, calcium inhibits PY at pH 7.8). These results show for the first time a role for calcium/calmodulin-dependent kinases in PRKA-dependent sperm PY; a non-PRKA-dependent pathway regulating sperm PY; and the apparent involvement of the FAK family of protein tyrosine kinases downstream in both pathways.
Wagner B, Ainsworth DM, Freer H.Soluble CD14 (sCD14) binds bacterial lipopolysaccharide (LPS) and acts as an anti-inflammatory LPS-inhibitor in vivo. In humans, sCD14 is one of the soluble biomarkers used for various inflammatory diseases and conditions, however, sCD14 assays have not yet been evaluated in horses. Here, we developed and optimized a bead-based assay for the quantification of sCD14 in horses. The assay was then used to determine native sCD14 concentrations in serum from healthy and septic foals, in the colostrum of healthy mares and in plasma from adult horses with recurrent airway obstruction (RAO) and contro...
Druart X, Rickard JP, Mactier S, Kohnke PL, Kershaw-Young CM, Bathgate R, Gibb Z, Crossett B, Tsikis G, Labas V, Harichaux G, Grupen CG, de Graaf SP.Seminal plasma contains a large protein component which has been implicated in the function, transit and survival of spermatozoa within the female reproductive tract. However, the identity of the majority of these proteins remains unknown and a direct comparison between the major domestic mammalian species has yet to be made. As such, the present study characterized and compared the seminal plasma proteomes of cattle, horse, sheep, pig, goat, camel and alpaca. GeLC-MS/MS and shotgun proteomic analysis by 2D-LC-MS/MS identified a total of 302 proteins in the seminal plasma of the chosen mammali...
Fontenot RL, Sink CA, Werre SR, Weinstein NM, Dahlgren LA.This study evaluated the quality and bacteriologic safety of platelet-rich plasma (PRP) produced by 3 simple, inexpensive tube centrifugation methods and a commercial system. Citrated equine blood collected from 26 normal horses was processed by 4 methods: blood collection tubes centrifuged at 1200 and 2000 × g, 50-mL conical tube, and a commercial system. White blood cell (WBC), red blood cell (RBC), and platelet counts and mean platelet volume (MPV) were determined for whole blood and PRP, and aerobic and anaerobic cultures were performed. Mean platelet concentrations ranged from 1.55- to 2...
Zylicz-Stachula A, Zolnierkiewicz O, Jasiecki J, Skowron PM.Genomics and metagenomics are currently leading research areas, with DNA sequences accumulating at an exponential rate. Although enormous advances in DNA sequencing technologies are taking place, progress is frequently limited by factors such as genomic contig assembly and generation of representative libraries. A number of DNA fragmentation methods, such as hydrodynamic sharing, sonication or DNase I fragmentation, have various drawbacks, including DNA damage, poor fragmentation control, irreproducibility and non-overlapping DNA segment representation. Improvements in these limited DNA scissi...
Donnelly KS, Giuliano EA, Sharma A, Tandon A, Rodier JT, Mohan RR.To explore (i) the potential of polyethylenimine (PEI) nanoparticles as a vector for delivering genes into equine corneal fibroblasts (ECFs) using green fluorescent protein (GFP) marker gene, (ii) whether PEI nanoparticle-mediated decorin (DCN) gene therapy could be used to inhibit fibrosis in the equine cornea using an in vitro model. Methods: Polyethylenimine-DNA nanoparticles were prepared at nitrogen-to-phosphate (N-P) ratio of 15 by mixing 22 kDa linear PEI and a plasmid encoding either GFP or DCN. ECFs were generated from donor corneas as previously described. Initially, GFP was introduc...
Wang CC, Hartmann-Fischbach P, Krueger TR, Wells TL, Feineman AR, Compton JC.Dermorphin and HYP(6) -dermorphin are hepta-peptides and natural opioids originally isolated from the skin of South American frogs. They are more potent than morphine but less likely to produce drug tolerance and addiction. These properties make them ideal candidates for the doping of racehorses to enhance performance during competition. Dermorphin was recently classified as a Class I drug by Racing Commissioners International (RCI), indicating that it is a banned substance in equine athletes. To enforce this ban, a fast and sensitive method was developed for dermorphin and HYP(6)-dermorphin a...
Yan W, Saleem MH, McDonough P, McDonough SP, Divers TJ, Chang YF.Leptospira immunoglobulin (Ig)-like (Lig) proteins are a novel family of surface-associated proteins in which the N-terminal 630 amino acids are conserved. In this study, we truncated the LigA conserved region into 7 fragments comprising the 1st to 3rd (LigACon1-3), 4th to 7.5th (LigACon4-7.5), 4th (LigACon4), 4.5th to 5.5th (LigACon4.5-5.5), 5.5th to 6.5th (LigACon5.5-6.5), 4th to 5th (LigACon4-5), and 6th to 7.5th (LigACon6-7.5) repeat domains. All 7 recombinant Lig proteins were screened using a slot-shaped dot blot assay for the diagnosis of equine leptospirosis. Our results showed that Li...
Klein C, Troedsson MH, Rutllant J.The process of water movement in the excurrent duct system of the male reproductive tract is pivotal for establishment of male fertility. The objective was to elucidate expression of aquaporin (AQP) water channels in the stallion reproductive tract. Real-time RT-PCR detected expression of AQP0-5 and AQP7-11 in testis, epididymis, and ductus deferens of mature stallions. There were two main expression patterns: (1) higher expression in testis than in epididymis and ductus deferens (AQP0, -4, -5, -8, -10, and -11); and (2) lower expression in testis than in epididymis and ductus deferens (AQP1, ...
Yadav SC, Kumar R, Kumar V, Jaideep , Kumar R, Gupta AK, Bera BC, Tatu U.Trypanosoma evansi is the most extensively distributed trypanosome responsible for disease called surra in livestock in many countries including frequent outbreaks in India. The prevalence of this disease is most commonly reported by standard parasitological detection methods (SPDM); however, antibody ELISA is being in practice by locally produced whole cell lysate (WCL) antigens in many countries. In the present investigation, we attempted to identify and purify immuno dominant, infection specific trypanosome antigens from T. evansi proteome using experimentally infected equine serum by immun...
Xie J, Liu G, Tian Z, Luo J.Several approaches have been developed for diagnosis of Theileria equi infection in horses and donkeys but all of them have limitations in practice. Due to numerous strengths including easy operation, cheapness and high sensitivity and specificity, LAMP has been already extensively used for surveillance of a number of diseases. We here set up a LAMP assay based on 18S rRNA gene for T. equi diagnosis. The approach was specific enough to differentiate T. equi from other evolutionary-related protozoa. Moreover, it was sensitive enough that LAMP was capable of detecting as much low as 10 copy targ...
Neuhauser S, Handler J.The aims of this study were to compare two different methods of quantifying the colour of the luminal surface of the equine endometrium and to relate the results to histopathological evidence of inflammation and fibrosis. The mucosal surfaces of 17 equine uteri obtained from an abattoir were assessed using a spectrophotometer and by computer-assisted analysis of photographs. Values were converted into L(*)a(*)b(*) colour space. Although there was significant correlation between the two methods of quantification, variations in 'brightness', 'red' and 'yellow' values were noted. Within a given u...
Seo JP, Tsuzuki N, Haneda S, Yamada K, Furuoka H, Tabata Y, Sasaki N.Mesenchymal stem cells (MSCs) are promising candidates for cell-based therapy and tissue engineering approaches. Fetal bovine serum (FBS) is commonly used for in vitro MSC expansion; however, the use of FBS may be associated with ethical, scientific, and safety issues. This study aimed to compare the ability of allogeneic platelet lysate (PL) and FBS to cause equine bone marrow-derived MSC expansion. MSCs were isolated from bone marrow aspirate in media supplemented with either PL or FBS, and cell proliferation properties and characteristics were examined. There were no significant differences...
Paillot R, Prowse L, Montesso F, Stewart B, Jordon L, Newton JR, Gilkerson JR.Equine influenza (EI) is a major respiratory disease of horses. Recent outbreaks of EI have demonstrated the ease with which EI virus (EIV) can be transmitted internationally. This study aimed to improve our understanding of EIV shedding after infection of vaccinated horses, which would inform possible changes to current quarantine requirements. Our objectives were to compare commonly used diagnostic tests and to evaluate the relative merits of nasal and nasopharyngeal swabs for detection of EIV in vaccinated and unvaccinated ponies following EIV infection and to use these data to inform optim...
Cordeiro Rde A, Bittencourt PV, Brilhante RS, Teixeira CE, Castelo-Branco Dde S, Silva ST, De Alencar LP, Souza ER, Bandeira Tde J, Monteiro AJ....Respiratory infections are a common problem among equines and occur with variable rates of morbidity and mortality. Although some fungal species are considered primary agents of respiratory tract infections in several mammals, their relevance in respiratory diseases of equines is frequently neglected. In the present study, we performed an active search for Candida spp. in the nasal cavity of horses. The presence of Candida spp. was investigated through the use of nasal swabs that were streaked on culture media. These yeasts were identified through physiological testing and their in vitro antif...
Blong AE, Epstein KL, Brainard BM.To investigate the in vitro effects of 3 hydroxyethyl starch (HES) solutions on viscoelastic coagulation testing and platelet function in horses. Methods: Blood samples collected from 7 healthy adult horses. Methods: Blood samples were diluted with various crystalloid and HES solutions to approximate the dilution of blood in vivo that occurs with administration of a 10 and 20 mL/kg fluid bolus to a horse (1:8 and 1:4 dilutions, respectively). Diluted samples were analyzed through optical platelet aggregometry, platelet function analysis, thromboelastography, and dynamic viscoelastic coagulomet...
Kisiday JD, Goodrich LR, McIlwraith CW, Frisbie DD.To compare the mesenchymal stem cell (MSC) yield and chondrogenic and osteogenic differentiation from 5- and 50-mL bone marrow aspirates from horses. Methods: Six 2- to 5-year-old mixed-breed horses. Procedures-2 sequential 5-mL aspirates were drawn from 1 ilium or sternebra. A single 50-mL aspirate was drawn from the contralateral ilium, and 2 sequential 50-mL aspirates were drawn from a second sternebra. The MSC yield was determined through the culture expansion process. Chondrogenesis and osteogenesis were evaluated by means of conventional laboratory methods. Results: The second of the 2 s...
Bailly-Chouriberry L, Cormant F, Garcia P, Kind A, Popot MA, Bonnaire Y.Cobra venom (Naja kaouthia) contains a toxin called α-cobratoxin (α-Cbtx). This toxin is a natural protein containing 71 amino acids (MW 7821 Da) with a reported analgesic potency greater than morphine. In 2007, in USA, this substance was found in the barns of a thoroughbred trainer and since then till date, the lack of a detection of this molecule has remained a recurring problem for the horseracing industry worldwide. To solve this problem, the first method for the detection of α-cobratoxin in equine plasma has now been developed. Plasma sample (3 mL) was treated with ammonium sulfate at ...
McFadden AM, Pearce PV, Orr D, Nicoll K, Rawdon TG, Pharo H, Stone M.To summarise investigation and laboratory data collected between 2001 and 2011 to provide evidence that equine arteritis virus is not present in the horse population of New Zealand. Methods: Analysis was carried out on results from laboratory tests carried out at the Ministry for Primary Industries Animal Health Laboratory (AHL) for equine arteritis virus from horses tested prior to being imported or exported, testing of stallions as part of the New Zealand equine viral arteritis (EVA) control scheme and testing as part of transboundary animal disease (TAD) investigations for exclusion of EVA....
Penrod LV, Allen RE, Rhoads ML, Limesand SW, Arns MJ.Uterine inflammation results in a poor uterine environment and early embryonic loss in the mare due to an inhibition of maternal recognition of pregnancy caused from increased prostaglandin F2α (PGF2α). Oxytocin binds to endometrial cell receptors to activate prostaglandin synthesis. An oxytocin receptor antagonist (Atosiban) and a cyclooxygenase inhibitor (indomethacin) both decrease PGF2α production. The aim of this study was to evaluate the in vitro effects of Atosiban and indomethacin on equine uterine prostaglandin secretion. Equine endometrial explants were harvested on day two of beh...
Guzeloglu A, Atli MO, Kurar E, Kayis SA, Handler J, Semacan A, Aslan S.The aims of the present study were to elucidate the expression profiles of leukotriene (LT) pathway mRNA transcription and to determine the possible interaction of LT and prostaglandin (PTG) pathways genes in equine endometrium during the estrous cycle and early pregnancy. Endometrial biopsies were obtained from mares on the day of ovulation (d0), at late diestrous (LD, n = 4), and after luteolysis in the estrus phase (AL, n = 4) of the cycle. Biopsies were also taken on Days 14 (P14; n = 4), 18 (P18, n = 4), and 22 (P22, n = 4) during early pregnancy that were comparable days to cyclic sampli...
Lygren T, Schjerling P, Jacobsen S, Berg LC, Nielsen MO, Langberg H, Thomsen PD.Insulin-like growth factor (IGF-1) is an important mediator of tissue repair in horses. Objective: The aim of the study was to evaluate whether IGF-1 could be measured reliably in equine serum and tendon tissue extracts, using an IGF-1 ELISA kit developed for human serum and plasma. Methods: A glycyl-glycine pretreatment protocol of samples was compared with the pretreatment procedure recommended by the manufacturer. Intra- and inter-assay imprecision were evaluated by repeated measurements of equine serum pools. Assay inaccuracy was determined based on the linearity of serially diluted equine...
Peters LW, Smiet E, de Sain-van der Velden MG, van der Kolk JH.In particular branched-chain amino acids might limit muscle protein loss in pathological conditions. Little is known on basic amino acid utilization of muscle in horses. Objective: To assess amino acid utilization by the hindlimb of horses at rest and following low intensity exercise. ANIMALS & METHODS: Amino acid uptake by the hindlimb was investigated using the arteriovenous difference technique. Blood from six warmblood mares (mean age 12 ± 3 (SD) years and weighing 538 ± 39 kg) was collected simultaneously from the (transverse) facial artery and from the caudal vena cava. Food was wi...
Oguma K, Ishida M, Maeda K, Sentsui H.Equine cells are required for isolation of viruses that infect the horse. However, only a few equine cell lines and cell cultures are available so far. Fetal horse kidney (FHK)-Tcl3.1 cell is a novel cell line established by introducing simian virus 40 (SV40) large T antigen. In the present study, the ability to propagate equine viruses was compared between FHK-Tcl3.1 cells and other equine cells. FHK-Tcl3.1 cells efficiently increased many viruses derived from or having pathogenicity to horses and produced high infective titers in culture fluids. These results indicate that FHK-Tcl3.1 cells w...
Morató R, Soares JM, Orero G, Mogas T, Miró J.The effect of combining double layer density gradient centrifugation (DL-DGC) with different capacitation treatments on the fertilising capacity of frozen-thawed stallion sperm was examined via a heterologous assay involving in vitro-matured, zona pellucida-free bovine oocytes. In a first experiment, aliquots of frozen-thawed stallion sperm were subjected to one of five capacitation treatments without DL-DGC - ionomycin at 1.0μM, 0.1μM, 0.05μM or 0.01μM, or caffeine at 200μg/mL. The fertilising capacity of the semen was then assessed at 18h by staining the above oocytes with 4,6-diamidino...
BMC research notesApril 16, 2013
Volume 6 152 doi: 10.1186/1756-0500-6-152
Christensen MB, Sørensen JC, Jacobsen S, Kjelgaard-Hansen M.Serum amyloid A (SAA) is useful as a diagnostic marker of systemic inflammation in horses, but only heterologous assays based on non-equine calibration and standardization are available for measurements of equine SAA. More accurate measurements could be obtained using purified species-specific SAA in native conformation for assay calibration and standardization. Further knowledge about the biochemical properties of SAA would facilitate a future production of native species-specific calibration material Therefore, the aim of the study was an investigation of the solubility and potentials for pu...
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 =...
Maischberger E, Cummins CA, Fitzpatrick E, Gallagher ME, Worrall S, Rousseau K, Thornton DJ, Meijer WG, Miranda-Casoluengo R, Duggan VE....In the equine reproductive tract, little is known about mucin gene expression and the role of mucins in barrier function and host-cell interaction. The aims of the study were to identify equine orthologs of mammalian mucin genes using available equine sequence data, to profile expression of equine orthologous mucin genes in the endometrium using reverse transcriptase polymerase chain reaction (RT-PCR), to determine spatial expression patterns of mucin genes using in situ hybridisation, and to confirm the presence of mucin gene products using Western blotting and equine-specific mucin antibodie...
Binder AK, Rodriguez KF, Hamilton KJ, Stockton PS, Reed CE, Korach KS.Determining the spatial and temporal expression of genes involved in the ovulatory pathway is critical for the understanding of the role of each estrogen receptor in the modulation of folliculogenesis and ovulation. Estrogen receptor (ER)-β is highly expressed in ovarian granulosa cells, and mice lacking ER-β are subfertile due to inefficient ovulation. Previous work has focused on isolated granulosa cells or cultured follicles and, although informative, provides confounding results due to the heterogeneous cell types present including granulosa and theca cells and oocytes and exposure to in...
Guan F, Uboh CE, Soma LR, Robinson M, Maylin GA, Li X.Dermorphin is a unique opioid peptide that is 30-40 times more potent than morphine. It was misused and went undetected in horse racing until 2011 when intelligence obtained from a few North American race tracks suggested its use. To prevent such misuse, a reliable analytical method became necessary for detection and identification of dermorphin in post-race horse samples. This paper describes the first liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for such a purpose. Equine plasma and urine samples were pre-treated with ethylenediamine tetra-acetic acid and urea prior to so...
Barta O, Oyekan PP.Lymphocyte transformation test is a powerful tool in laboratory testing of immunologic competence of animals. The impaired function of the lymphocytes or presence of mitogenesis suppressing factors in the patient serum were detected by comparing lymphocyte transformation (expressed as thymidine incorporation) obtained in media containing either autologous, homologous, or fetal calf serum additions. Most valuable results were obtained by using at least two, preferably three, different phytomitogens: concanavalin A (Con A), pokeweed mitogen (PWM), and phytohemagglutinin (PHA) at optimal concentr...
Thway TM, Clay CM, Maher JK, Reed DK, McDowell KJ, Antczak DF, Eckert RL, Nilson JH, Wolfe MW.Immortalized cell lines have many potential experimental applications including the analysis of molecular mechanisms underlying cell-specific gene expression. We have utilized a recombinant retrovirus encoding the simian virus-40 (SV-40) large T antigen to construct several immortalized cell lines of equine chorionic girdle cell lineage - the progenitor cells that differentiate into the equine chorionic gonadotropin (eCG) producing endometrial cups. Morphologically, the immortalized cell lines appear similar to normal chorionic girdle cells. Derivation of the immortalized cell lines from a cho...
Sharma AK, Rajashankar KR, Yadav MP, Singh TP.The structure of mare apolactoferrin (MALT) has been determined at 3. 8 A resolution by the molecular-replacement method, using the structure of mare diferric lactoferrin (MDLT) as the search model. The MDLT structure contains two iron-binding sites: one in the N-terminal lobe, lying between domains N1 and N2, and one in the C-terminal lobe between domains C1 and C2. Both lobes have a closed structure. MALT was crystallized using the microdialysis method with 10%(v/v) ethanol in 0.01 M Tris-HCl. The structure has been refined to a final R factor of 0.20 for all data to 3.8 A resolution. Compar...
Morais JF, de Freitas MC, Yamaguchi IK, dos Santos MC, da Silva WD.IgG and F(ab')2 fragments were prepared from horse plasma rich in specific antibodies against Brazilian Bothrops or Crotalus venoms. Both preparations, free of gross contamination with non-immunoglobulin proteins, were able to combine in vitro with their respective antigens, forming immune complexes at antigen excess, equivalence or antibody excess, and activating the C system, through either the classical or the alternative pathways. The IgG preparation was more effective in neutralizing the lethal factors in Bothrops or Crotalus venoms, compared with the F(ab')2 fragments. In contrast, IgG a...
Ito M, Itou T, Sakai T, Santos MF, Arai YT, Takasaki T, Kurane I, Ito FH.Brain samples from different animal species including humans: five vampire bats, 14 cattle, 12 dogs, 11 cats, two horses, one pig, one sheep and three humans collected from various geographical regions of Brazil were found to be positive for rabies by means of the fluorescent antibody test (FAT) and the mouse inoculation test (MIT). The brain samples were retested for rabies by means of the reverse transcription and polymerase chain reaction (RT-PCR) with 2 primer sets (P1/P2 and RHNI/RHNS3), which amplified full or partial regions on the nucleoprotein (N) gene of the rabies virus, respectivel...
Charvat RA, Breitenbach JE, Ahn B, Zhang Y, O'Callaghan DJ.Defective interfering particles (DIP) of equine herpesvirus 1 (EHV-1) inhibit standard virus replication and mediate persistent infection. The DIP genome is comprised of only three genes: UL3, UL4, and a hybrid gene composed of portions of the IR4 (EICP22) and UL5 (EICP27) genes. The hybrid gene is important for DIP interference, but the function(s) of the UL3 and UL4 genes are unknown. Here, we show that UL4 is an early gene activated solely by the immediate early protein. The UL4 protein (UL4P) was detected at 4hours post-infection, was localized throughout the nucleus and cytoplasm, and was...
Witonsky S, Gogal RM, Buechner-Maxwell V, Ahmed SA.To determine whether immune function can be accurately assessed in blood samples obtained from horses and refrigerated overnight and whether a nonradioactive lymphocyte proliferation assay can be used to evaluate samples obtained from horses. Methods: 224 blood samples from 28 clinically normal adult horses. Methods: Heparinized blood samples were collected. Each sample was divided into 2 equal aliquots. One aliquot was refrigerated overnight to simulate overnight shipping of blood samples, and the other aliquot was evaluated on the day of blood collection. Lymphocytes were isolated and enumer...
Liu L, Zhang Y, Ma H, Cao H, Liu W.Horse Milk has important quantitative characteristics and high economic value. However, the DNA methylation regulators involved in horse milk traits have not been clarified. To explore the important role of genome-wide DNA methylation in regulating equine milk yield, this study systematically investigated the genome-wide DNA methylation profiles of Kazakh horse blood by comparing a high-production group (HP, average daily milk yield of 7.5 kg) and low-production group (LP, average daily milk yield of 3.2 kg) using deep whole-genome bisulfite sequencing. First, both groups showed similar prop...
Lauder RM, Huckerby TN, Nieduszynski IA.Fibromodulin has been isolated from bovine and equine articular cartilage and the attached keratan sulphate chains subjected to digestion by keratanase II. The oligosaccharides generated have been reduced and subsequently isolated by strong anion-exchange chromatography. Their structures have been determined by high-field 1H-NMR spectroscopy and high-pH anion-exchange chromatography. Both alpha(2-6)- and alpha(2-3)-linked N-acetylneuraminic acid have been found in the capping oligosaccharides, and, fucose which is alpha(1-3)-linked to N-acetylglucosamine has been found as a branch in both repe...
Ramery E, Fraipont A, Richard EA, Art T, Pirottin D, van Delm W, Bureau F, Lekeux P.Inflammatory airway disease (IAD) affects performance and well-being of horses. Diagnosis is primarily reached by bronchoalveolar lavage (BAL) cytology which is invasive and requires sedation. Objective: The purpose of this study was to identify differential gene expression in peripheral blood of horses with IAD using species-specific expression microarrays. Methods: Equine gene expression microarrays were used to investigate global mRNA expression in circulating leukocytes from healthy, IAD-affected, and low-performing Standardbred and endurance horses. Results: Nine genes in Standardbred and...
Scocco P, Pedini V.Sialic acids modulate the metabolite transport across membranes and may be involved in protection against pathogenic agents. The presence of sialoderivatives in the equine mandibular gland requires further study. Objective: To biochemically visualise in situ the presence of sialoderivatives, by means of mild and strong periodate oxidation and alcoholic saponification, combined with lectin histochemistry and sialidase digestion in order to hypothesise roles for detached sialoderivatives. Methods: Mandibular glands were removed from 8 mature horses of both sexes and subjected to histochemical pr...
Dell'aquila ME, Fusco S, Lacalandra GM, Maritato F.The aim of this study was to develope an efficient and reproducible procedure for in vitro maturation (IVM) and fertilization (IVF) in the horse. Cumulus-oocyte complexes (COCs) recovered from the ovaries of mares slaughtered during the breeding season were morphologically evaluated, and those showing a compact cumulus and homogeneously appearing cytoplasm were selected for culture. Effects on the maturation of estrous mare serum (EMS) versus estrous cow serum (ECS) as medium supplement were also evaluated (Experiment 1). In Experiment 2, the fertilization of in vitro matured oocytes with froz...
Perelygin AA, Lear TL, Zharkikh AA, Brinton MA.Mammalian 2'-5' oligoadenylate (2-5A) synthetases are important mediators of the antiviral activity of interferons. Both human and mouse 2-5A synthetase gene families encode four forms of enzymes: small, medium, large and ubiquitin-like. In this study, the structures of four equine OAS genes were determined using DNA sequences derived from fifteen cDNA and four BAC clones. Composition of the equine OAS gene family is more similar to that of the human OAS family than the mouse Oas family. Two OAS-containing bovine BAC clones were identified in GenBank. Both equine and bovine BAC clones were phy...
Dube F, Hinas A, Roy S, Martin F, Åbrink M, Svärd S, Tydén E.The nematode Parascaris univalens is one of the most prevalent parasitic pathogens infecting horses but anthelmintic resistance undermines treatment approaches. The molecular mechanisms underlying drug activity and resistance remain poorly understood in this parasite since experimental in vitro models are lacking. The aim of this study was to evaluate the use of Caenorhabditis elegans as a model for P. univalens drug metabolism/resistance studies by a comparative gene expression approach after in vitro exposure to the anthelmintic drug ivermectin (IVM). Methods: Twelve adult P. univalens worms...
Godard S, Vaiman A, Schibler L, Mariat D, Vaiman D, Cribiu EP, Guérin G.The purpose of this study was to increase the number of genes assigned by in situ hybridization to equine chromosomes and thus the number of links for comparative mapping with other species. Forty-four new sequences were added to the horse cytogenetic map by FISH mapping of BAC clones containing genes (35) or ESTs (9). Three approaches were developed: use of horse BAC clones screened with (i) horse EST primers, (ii) interspecific consensus intraexonic primers, and (iii) use of goat BAC containing genes previously localized on goat chromosomes. Present data suggest that the second approach is t...
Mengual R, Leblanc G, Sudaka P.The present study determines the characteristics of isotopic Na and lactate exchange under equilibrium conditions in horse kidney brush-border membrane vesicles. The influence of one solute (Na+ or lactate) on the isotopic exchange of the co-transported species (lactate or Na) was analyzed in detail. Analysis of the data suggests that Na and lactate interact sequentially with the carrier. The observed apparent symmetry between the activating effect of low Na concentrations and the inhibiting effect of high Na concentrations on the lactate exchange process suggests that the carrier functions ac...
Schlupp A, Anielski P, Thieme D, Müller RK, Meyer H, Ellendorff F.The beta2-agonist clenbuterol is commonly administered for therapeutic purposes in the horse, but its use an an anabolic agent is illegal. Clenbuterol can be detected in blood and urine for a relatively short period after administration and detection in hair could enhance the analytical range and be used to determine the history of clenbuterol application. Objective: That detection in mane or tail hair is possible over an extended period. Methods: Four horses received 0.8 microg clenbuterol hydrochloride/kg bwt b.i.d. for 10 days. Four other horses were used as untreated controls. Blood, urine...
Xiao C, Liu Y, Jiang Y, Magoffin DE, Guo H, Xuan H, Wang G, Wang LF, Tu C.Four monoclonal antibodies (mAbs) were generated by immunizing BALB/C mice with recombinant nucleocapsid protein (N) of Nipah virus (NiV) and Hendra virus (HeV) expressed in E. coli. Two mAbs each were obtained for the HeV N and NiV N, respectively. All four mAbs displayed specific reactivity with the recombinant N proteins of both viruses by western blot, which was further confirmed by immunofluorescent antibody assay using fixed insect cells infected with recombinant baculoviruses expressing either the HeV or NiV N protein. Epitope mapping using a 12-mer random peptide phage display library ...
Echigoya Y, Sato T, Itou T, Endo H, Sakai T.The species-specific properties of LDH isozymes are essentially determined by M (muscle) and H (heart) subunit proteins encoded by the LDHA and LDHB genes, respectively. In the present study, we molecularly characterized the full-length equine lactate dehydrogenase A (eLDHA) and B (eLDHB) cDNAs. The eLDHA cDNA consisted of a 999-bp open reading frame (ORF), while the eLDHB and newly acquired bat LDHB consisted of a 1002-bp ORF, which is 3 bp shorter than the LDHB ORF of other registered mammals. The alignment of amino acid sequences showed that eLDHA acquired positively charged His 88 and 226,...
Guan M, Dai D, Li L, Wei J, Yang H, Li S, Zhang Y, Lin Y, Xiong S, Zhao Z.High levels of triacylglycerols (TGs) have been linked to cardiovascular disease and liver diseases. Comprehensively analyzing TGs is helpful to understand the TGs functions in these diseases. However, due to the existence of a large number of isomers TGs and the lack of commercial standards, precise analysis of individual triacylglycerol (TG) with specific fatty acid chain composition is full of challenge. In this work, ultra-performance liquid chromatography (UPLC) coupled with electrospray ionization (ESI) mass spectrometry (MS) were employed for comprehensive qualification and quantificati...
Cardier J, Romano E, Soyano A.Iron and ferritin impair a variety of immunological functions. To evaluate the effect of ferritin iron content on rat lymphocyte proliferative response, isoferritins that differ in their iron content and isoelectric point (pI) were isolated from iron overload rat livers by ultracentrifugation (isoferritins with high iron content and low pI) or crystallization (isoferritins with low iron content and high pI) methods. Additionally, commercial horse splenic ferritin (with a lower pI and higher iron content than rat isoferritins) was also tested. Proliferative response to Con A was decreased in a ...
Hitchcock PJ, Brown TM, Corwin D, Hayes SF, Olszewski A, Todd WJ.Examination of recently isolated cultures of three strains of Contagious Equine Metritis Organism grown on specially formulated, serum-free, clear typing medium revealed the presence of numerous colonial opacity variants. These colonies were prepared by a number of fixation and staining techniques and examined by scanning and transmission electron microscopy. Opaque and transparent phenotypes produced copious amounts of extracellular material compared with intermediate-opacity phenotypes which produced little or none. Also unique to intermediate colonies were numerous thin intercellular strand...
Wang LF, Gould AR, Selleck PW.Full-length cDNA clones coding for the matrix (M) and fusion (F) proteins of equine morbillivirus (EMV) were isolated by RT-PCR, and expressed in Escherichia coli using two different expression systems. Western blot analysis indicated that the M and F proteins, expressed either by itself or as fusion proteins with glutathione S-transferase (GST), were insoluble and degraded after expression. Analysis of the degradation pattern of recombinant M protein suggested that the N-terminus of the matrix protein might be more stable and antigenic than the C-terminal region. Therefore a third system was ...
Bauer N, Moritz A.Besides flow cytometric detection of cellular hemoglobin (HGB) concentration, the ADVIA 2120 uses a novel cyanide-free colorimetric method to determine extracellular total HGB concentration. In human samples, the results are equivalent to those of the cyanmethemoglobin method on the ADVIA 120. Cyanide-free HGB measurement has not been evaluated in animal samples. Objective: The aim of this prospective study was to compare the 3 methods of HGB analysis on the ADVIA 2120 and ADVIA 120 in blood samples from dogs, cats, and horses. Methods: Consecutive fresh K(3)EDTA blood samples from 119 dogs, 1...
Thaker SR, Dutta SK, Adhya SL, Mattingly-Napier BL.A gene bank of Ehrlichia risticii was constructed in plasmid vector pUC13. Five clones representing discrete regions of the E. risticii genome were tested for their ability to hybridize specifically to E. risticii DNA. None of the clones cross-hybridized with Ehrlichia equi DNA, whereas four of these clones cross-hybridized with Ehrlichia canis and Ehrlichia sennetsu DNAs. However, one clone carrying a 1-kilobase HindIII fragment of E. risticii DNA failed to cross-react with the genomes of E. sennetsu, E. canis, and E. equi in dot blot hybridization assays. The sensitivity of this probe for th...
Petry S, Sévin C, Kozak S, Foucher N, Laugier C, Linster M, Breuil MF, Dupuis MC, Hans A, Duquesne F, Tapprest J.Study of the rifampicin resistance of Rhodococcus equi strains isolated from French horses over a 20-year period. Rifampicin susceptibility was tested by disk diffusion (DD) and broth macrodilution methods, and rpoB gene sequencing and MLST were performed on 40 R. equi strains, 50.0% of which were non-susceptible to rifampicin. Consistency of results was observed between rifampicin susceptibility testing and rpoB sequencing. Strains non-susceptible to rifampicin by DD had a substitution at one of the sites (Asp516, His526 and Ser531) frequently encountered and conferring rifampicin resistance....
Fernández-Pinero J, Fernández-Pacheco P, Rodríguez B, Sotelo E, Robles A, Arias M, Sánchez-Vizcaíno JM.A highly sensitive and specific TaqMan-MGB real-time RT-PCR assay has been developed and standardised for the detection of African horse sickness virus (AHSV). Primers and MGB probe specific for AHSV were selected within a highly conserved region of genome segment 7. The robustness and general application of the diagnostic method were verified by the detection of 12 AHSV isolates from all of the nine serotypes. The analytical sensitivity ranged from 0.001 to 0.15 TCID(50) per reaction, depending on the viral serotype. Real-time PCR performance was preliminarily assessed by analysing a panel of...
Assunção P, Barbosa T, Yonezawa L, Barbosa L, Watanabe M, Kohayagawa A, Schmidt E.High-intensity exercise can be associated with the occurrence of muscle injury, as well as the induction of an acute-phase response (APR). The present study aims to investigate the synthesis and profile of serum proteins in horses before and after participating in 2 different exercise protocols and to relate this profile to the presence or absence of muscular injury caused by exercise. Ten purebred Arabian (n = 5) and Criollo (n = 5) horses were subjected to 2 different tests on a treadmill, one consisting of short-duration and rapid-acceleration training (TRA) that was mostly anerobic and the...
Merediz EF, Dyer J, Salmon KS, Shirazi-Beechey SP.Fructose can be a suitable carbohydrate supplement for horses before and/or during endurance exercise. In comparison to glucose, the ingestion of fructose results in a lower insulin peak and less marked fluctuations in blood glucose during exercise, potentially avoiding hypoglycaemia-induced exhaustion. Objective: To assess the capacity of the equine small intestine to absorb fructose and to determine the mechanism, molecular structure and properties of equine intestinal fructose transport. Methods: Using PCR-based strategies, RNA isolated from equine small intestine and primers designed to ho...
Smith R, Önnerfjord P, Holmgren K, di Grado S, Dudhia J.The diagnosis of tendon injury relies on clinical signs and diagnostic imaging but imaging is subjective and does not always correlate with clinical signs. A molecular marker would potentially offer a sensitive and specific diagnostic tool that could also provide objective assessment of healing for the comparison of different treatments. Cartilage Oligomeric Matrix Protein (COMP) has been used as a molecular marker for osteoarthritis in humans and horses but assays for the protein in tendon sheath synovial fluids have shown overlap between horses affected by tendinopathy and controls. We hypot...
