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.
Liu CC, Hsiao YC, Chu LJ, Wang PJ, Liu CH, Hsieh WC, Yu JS., also known as Taiwanese cobra, is one of the most prevalent venomous snakes in Taiwan. Clinically, freeze-dried neurotoxic antivenom (FNAV) produced from horses by Taiwan Centers for Disease Control (CDC) has been the only approved treatment for envenoming for the last few decades. During antivenom production, large numbers of mice are used in the in vivo assay to determine whether the neutralization potency of hyperimmunized equines is satisfactory for large-scale harvesting. However, this in vivo assay is extremely laborious, expensive, and significantly impairs animal welfare. In the pre...
Delcourt V, Garcia P, Pottier I, Mansoibou N, Bache N, Glavieux Y, Chabot B, Perot I, André F, Loup B, Barnabé A, Popot MA, Bailly-Chouriberry L.Synthetic androgenic anabolic steroids (AAS) are banned compounds and considered as major threats by both racing and sports international authorities. Hence, doping control laboratories are continually looking into analytical improvements to increase their detection capabilities, notably by means of emerging technologies. To enhance analytical performances for the detection of synthetic AAS such as stanozolol, specific chromatographic procedures have been developed using recent quaternary liquid chromatography technology originally designed for high-throughput standardized proteomics connected...
Giannetto C, Acri G, Giudice E, Arfuso F, Testagrossa B, Piccione G.Raman spectroscopy is an inelastic light-scattering phenomenon that provides vibrational spectrum that contains information relative to chemical bonds and symmetry of a specific molecule, allowing the quali-quantitative simultaneous determination of several components in the biological fluids. Raman spectroscopy measurement returns a spectrum over a wavenumber range constituted by several bands representing biomarkers according to investigated biological matrices. In literature, it has been reported that at the frequencies inside the (1,300-1,360) cm total lipids, and tryptophan have been iden...
Flick M, Vinther AML, Jacobsen S, Berg LC, Gimeno M, Verwilghen D, Howden W, Averay K, van Galen G.Serum neutrophil gelatinase-associated lipocalin (sNGAL) is a marker of renal injury, and its concentrations are affected by inflammation. Therefore, it could serve as a useful biomarker of disease or fitness in high-level competition. However, it has not yet been determined if sNGAL concentrations are affected by exercise. The aim of this study was to determine whether concentrations of equine sNGAL were affected by 1000 m galloping as the form of exercise used in the study. Pre- and post-gallop sNGAL, serum amyloid A, and creatinine concentrations were evaluated in 14 healthy Thoroughbred r...
Klett D, Combarnous Y.In previous studies, we had shown the synergistic effect of 10 M forskolin (FSK) on the detection threshold of the cyclic AMP response to luteinizing hormones (LH) and chorionic gonadotropins (CG) from various species in the mouse Leydig tumor cell (mLTC) cell line. Independently, we started to study the effect of 10-10 M oxytocin (OXT) also on the cyclic AMP response to LH and CG preparations on these same cells and found an amplifying effect on the luminescence response caused by gonadotropins. The aim was then to explore the effects of 10-10 M OXT on the gonadotropin-induced cAMP response, ...
Kiemle J, Hindenberg S, Bauer N, Roecken M.Rapid, accurate detection of serum amyloid A (SAA) is needed in equine practice. We validated a patient-side point-of-care (POC) assay (Stablelab; Zoetis) compared to the turbidimetric immunoassays LZ-SAA (TIA-Hum) and VET-SAA (TIA-Vet; both Eiken Chemical). Analytical performance was assessed at 3 different concentration ranges and with interferences. Inter-method comparison using 49 equine serum samples revealed a significant difference between median SAA results ( < 0.0001), with the strongest bias between the POC and TIA-Vet (median 1,093 vs. 578 mg/L). The median SAA value obtain...
Bozorgmanesh R, Thornton J, Snyder J, Fletcher C, Mack R, Coyne M, Murphy R, Hegarty E, Slovis N.Symmetric dimethylarginine (SDMA) is a renal biomarker correlated with glomerular filtration rate (GFR). Objective: Describe changes in SDMA in clinically healthy foals and their mares during the first month postfoaling. Methods: Convenience sampling of healthy periparturient Thoroughbred mares and their full-term foals from a population of client-owned horses. Methods: Serum and EDTA whole blood samples were collected from mares in their last month of pregnancy and then from mares and foals at approximately <12 hours, 48 hours, 7 days, and 30 days postbirth. Samples were processed ...
Ganeshpurkar A, Singh R, Kumar D, Gutti G, Sardana D, Shivhare S, Singh RB, Kumar A, Singh SK.Machine learning (ML), an emerging field in drug design, has the potential to predict toxicity, shape-based analysis of inhibitors, scoring function (SF) etc. In the present study, a homology model, docking protocol, and a dedicated SF have been developed to identify the inhibitors of horse butyrylcholinesterase (BChE) enzyme. Horse BChE enzyme has homology with human BChE and is a substitute for the screening of inhibitors. The developed homology model was validated and the active site residues were identified from Cavityplus to generate grid box for docking. The validation of docking invol...
Gong L, Shi F, Su S, Xie Q, Xian R, Hang B, Zhao Y.In recent years, due to the shortage of donkey skin resources, the price of Asini Corii Colla has seen a rapid increase. Consequently, fake gelatin prepared from horse, mules, pig, and cow skin has appeared in the market, resulting in unreliable quality of Asini Corii Colla. Therefore, there is an urgent need to develop an efficient and accurate method for improving the quality of Asini Corii Colla. Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was used to determine the donkey skin components in Asini Corii Colla. Accordingly, 0. l g of the evenly mixed sa...
Marčeková P, Mad'ar M, Styková E, Kačírová J, Sondorová M, Mudroň P, Žert Z.Equine hoof canker and bovine digital dermatitis are infectious inflammatory diseases of the hooves with an unknown etiology. However, anaerobic spirochetes of the genus Treponema are considered to be potential etiological agents. The aim of this study was to find a suitable way to isolate DNA and to detect the presence of treponemal DNA in samples of equine hoof canker and bovine digital dermatitis. DNAzol®® Direct and column kits were used to isolate DNA from samples of equine hoof canker and bovine digital dermatitis. The presence of Treponema spp. was detected using PCR and Sanger sequen...
Pusterla N, James K, Barnum S, Delwart E.Three newly identified equine parvoviruses (equine parvovirus hepatitis (EqPV-H), equine parvovirus CSF (EqPV-CSF) and equine copivirus (Eqcopivirus)) have recently been discovered in horses with respiratory signs. However, the clinical impact of these three equine parvoviruses has yet to be determined. Nasal fluid samples and blood from 667 equids with acute onset of fever and respiratory signs submitted to a diagnostic laboratory were analyzed for the presence of common equine respiratory pathogens (equine influenza virus, equine herpesvirus-1/-4, equine rhinitis A and B virus, subspecies )...
Hansson E, Skiöldebrand E.To treat osteoarthritic chondrocytes and thereby reduce the inflammation with a drug combination that primarily affects 5-HT- and ATP-evoked Ca2+ signaling. In osteoarthritic chondrocytes, Ca2+ signaling is elevated, resulting in increased production of ATP and inflammatory mediators. The expression of TLR4 and Na+/K+-ATPase was used to evaluate the inflammatory status of the cells. Methods: Equine chondrocytes were collected from joints with mild structural osteoarthritic changes and cultured in monolayers. The cells were treated with a combination of bupivacaine (1 pM) and sildenafil (1 ...
Tozaki T, Ohnuma A, Hamilton NA, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Nagata SI.Gene doping is prohibited for fair competition in human and horse sports. One style of gene doping is the administration of an exogeneous gene, called a transgene, to postnatal humans and horses. Although many transgene detection methods based on quantitative polymerase chain reaction (PCR), including real-time PCR and digital PCR, have been recently developed, it remains difficult to reliably detect low-copy transgenes. In this study, we developed and validated a nested digital PCR method to specifically detect low-copy transgenes. The nested digital PCR consists of (1) preamplification using...
Suchan T, Kusliy MA, Khan N, Chauvey L, Tonasso-Calvière L, Schiavinato S, Southon J, Keller M, Kitagawa K, Krause J, Bessudnov AN, Bessudnov AA....DNA hybridization-capture techniques allow researchers to focus their sequencing efforts on preselected genomic regions. This feature is especially useful when analysing ancient DNA (aDNA) extracts, which are often dominated by exogenous environmental sources. Here, we assessed, for the first time, the performance of hyRAD as an inexpensive and design-free alternative to commercial capture protocols to obtain authentic aDNA data from osseous remains. HyRAD relies on double enzymatic restriction of fresh DNA extracts to produce RNA probes that cover only a fraction of the genome and can serve a...
Geiger T, Gerhards H, Wollanke B.Equine recurrent uveitis (ERU) is typically caused by chronic intraocular leptospiral infection in warm-blooded horses in central Europe. The most effective therapy for leptospiral-induced ERU is the surgical removal of diseased vitreous (vitrectomy). Since vitrectomy is a highly specialized and invasive surgery, the indication must be determined very carefully. In order to obtain evidence of intraocular leptospiral infection by laboratory diagnostics in questionable leptospiral ERU-cases, sampling of aqueous humor is required, because serum tests using microscopic agglutination test (MAT) are...
Andrade DGA, Cerri FM, Barbosa GVM, Basso RM, Takahira RK, Pantoja JCF, Oliveira-Filho JP, Borges AS.Analysis of the cerebrospinal fluid (CSF) is important as a complementary test in horses with neurologic diseases, and sequential analysis may provide information about the treatment response or evolution and quantitative measures of the CSF drug concentration during treatment. The aim of this study was to compare erythrocyte and nucleated cell counts and protein concentration in multiple CSF samples obtained sequentially from two different puncture sites in clinically healthy horses. Eight and 12 horses, with no evidence of neurologic disease, were subjected to CSF collection from the atlanto...
Gaitskell-Phillips G, Martín-Cano FE, Ortiz-Rodríguez JM, da Silva-Álvarez E, Masot J, Redondo E, Gil MC, Ortega-Ferrusola C, Peña FJ.Seminal plasma proteins have important roles in sperm functionality, and different mechanisms including micro-vesicle transport of proteins are involved in the regulation of sperm biology. Due to the role of seminal plasma, we hypothesized that specific proteins present in seminal plasma may be used as discriminant variables with potential to identify stallions producing different quality ejaculates; 10 fertile stallions, with different motility and velocity values (although within normal ranges) were used in this study. Motilities and velocities were studied using computer assisted sperm anal...
Pearson G, Goodale M, Wakshlag J, Fortier L.The objective of this study was to determine the effects of an oral, commercially available, marine based omega-3 fatty acid (n3-FA) supplement on fatty acid characteristics in horse whole blood. Fifty healthy, adult horses of various light breeds were assigned to one of two treatment groups: Group 1 receiving 7.5g/day of the test supplement, and Group 2 receiving 15g/day of the test supplement. The supplement contained 0.092g docosahexaenoic acid (DHA) and 0.148g eicosapentaenoic acid (EPA) per gram. Therefore, Group 1 received 1.11g of EPA and 0.69g of DHA daily, while Group 2 received 2.22g...
Armando F, Mecocci S, Orlandi V, Porcellato I, Cappelli K, Mechelli L, Brachelente C, Pepe M, Gialletti R, Ghelardi A, Passeri B, Razzuoli E.Equine penile squamous cell carcinoma (epSCC) is the most frequent tumor of the external male genitalia, representing 67.5% of equine genital cancers. epSCC is associated with papilloma virus (PV) infection and has been recently proposed as a model for human PV-induced squamous cell carcinomas. It has already been suggested that epSCC might undergo epithelial-to-mesenchymal transition (EMT). This work aims to investigate in detail this process and the possible role of PV oncoproteins in epSCC. For this purpose, 18 penile SCCs were retrospectively selected and tested for both EcPV2 presence and...
McGlennon A, Waller A, Verheyen K, Slater J, Grewar J, Aanensen D, Newton R.Previously national surveillance data for monitoring strangles (Streptococcus equi infection) in UK horses was limited. Improved awareness and knowledge of positive diagnoses would permit the optimisation of biosecurity protocols, decreasing the prevalence of strangles. Methods: Seven UK laboratories reported positive strangles diagnoses between 1 January 2015 and 31 December 2019 based on identifying Streptococcus equi via agent detection assays from field-based practitioner-submitted samples. Associated clinical history and animal signalment were collected where provided, and descriptive ana...
Abdullah HHAM, Elbayoumy MK, Allam AM, Ashry HM, Abdel-Shafy S.Vector-borne bacterial diseases (VBBD) are a diverse group of tropical and subtropical zoonotic diseases. This study investigated the possibility of domestic animals to carry certain vector-borne bacterial microorganisms (VBBMs), as well as the presence of these targeted DNAs in their ectoparasites in different localities of Egypt using molecular analyses. For this study, 234 animal hosts (112 cattle, 38 sheep, 28 goats, 26 buffaloes, 22 donkeys, and 8 horses) in addition to 115 ectoparasites (95 ticks and 20 lice) were investigated for the molecular detection of Bartonella spp., Borrelia spp....
Munday JS, Gedye K, Daudt C, Chaves Da Silva F.Bovine papillomavirus (BPV) types 1 and 2 are recognized as the main cause of equine sarcoids. However, some studies report that up to a quarter of these tumors do not contain detectible BPV1 or BPV2 DNA. The absence of detectible BPV1 or BPV2 in these sarcoids suggests the possible involvement of other papillomavirus types. Currently, five are recognized to cause mesenchymal neoplasia after cross-species infection. In addition to BPV1 and BPV2, BPV13 has been associated with equine sarcoids in Brazil, BPV14 has been associated with feline sarcoids, and Ovis aries papillomavirus 2 caused a sa...
Moeller BC, Flores L, Clifford A, Alarcio G, Mosburg M, Arthur RM.Methylphenidate is a powerful central nervous system stimulant with a high potential for abuse in horse racing. The detection of methylphenidate use is of interest to horse racing authorities for both prior to and during competition. The use of hair as an alternative sampling matrix for equine anti-doping has increased as the number of detectable compounds has expanded. Our laboratory developed a liquid chromatography-high-resolution mass spectrometry method to detect the presence of methylphenidate in submitted samples. Briefly, hair was decontaminated, cut, and pulverized prior to liquid-liq...
Serafini R, Varner DD, Love CC.Flow cytometry procedures can be used for evaluation of both spermatogenic efficiency and diagnose disorders of stallion spermatogenesis. Aims of this study were to compare two testicular sample acquisition techniques (needle aspirate-N and tissue wedge-T) and results when using flow cytometry and histology procedures. Testicular cell types were stained with acridine orange, and nine regions (R2 to R10) were identified and enumerated following acquisition by either N or T. Testes were also grouped and analyzed by size and sexual maturity (Small [immature] compared with Large [mature]) and used...
Wehrman RF, Genschel U, Charli A, Kanthasamy AG, Allbaugh RA, Ben-Shlomo G.To establish a physiologically relevant ex vivo model of equine corneal epithelial wound healing. Methods: Fourteen equine corneas were randomly assigned to one of two groups: wounded (n = 8) or unwounded (n = 6) controls. In the wounded group, the axial corneal epithelium was removed by applying a 6 mm filter paper disk soaked in 1N-NaOH for 60 s. Corneas were subsequently cultured using an air-liquid interface model. Evaluation of corneal healing was performed daily, and culture medium was collected. Corneas were randomly assigned to undergo processing via histopathology and RNAscope i...
Bundgaard L, Åhrman E, Malmström J, Auf dem Keller U, Walters M, Jacobsen S.The objectives of this study was to establish a sensitive and reproducible method to map the cartilage and subchondral bone proteomes in quantitative terms, and mine the proteomes for proteins of particular interest in the pathogenesis of osteoarthritis (OA). The horse was used as a model animal. Protein was extracted from articular cartilage and subchondral bone samples from three horses in triplicate by pressure cycling technology or ultrasonication. Digested proteins were analysed by data independent acquisition based mass spectrometry. Data was processed using a pre-established spectral li...
Hifumi T, Akioka K, Tanaka T, Miyoshi N.Alveolar echinococcosis, which is caused by a larval-stage infection of Echinococcus multilocularis, is a zoonosis with public health importance. Recently, alveolar echinococcosis in slaughtered horses has been reported in Japan and Poland. In terms of public health, a highly sensitive and specific diagnostic method is essential for early detection during meat inspection. In this study, the loop-mediated isothermal amplification (LAMP) assay was developed and validated to target the mitochondrial cytochrome b (cob) gene of E. multilocularis. Forty-one hepatic solid nodules obtained from each h...
Tozaki T, Kwak HG, Nakamura K, Takasu M, Ishii H, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Hirata M, Nirasawa T, Nagata SI.In human and equestrian sporting events, one method of gene doping is the illegal use of therapeutic oligonucleotides to alter gene expression. In this study, we aimed to identify therapeutic oligonucleotides via sequencing using matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). As a model of therapeutic oligonucleotides, 22 bp-long phosphorothioated oligonucleotides (PSOs) were used. By using a Clarity OTX kit for extracting short-length oligonucleotides, a spectrum of singly charged PSO with a mean intensity of 6.08 × 10 (standard deviation: ...
Conze TM, Bagó Z, Revilla-Fernández S, Schlegel J, Goehring LS, Matiasek K.A final diagnosis in a horse with clinical signs of encephalopathy can be challenging despite the use of extensive diagnostics. Clinical signs are often not pathognomonic and need to be interpreted in combination with (specific) laboratory results and epidemiological data of the geographical region of the origin of the case(s). Here we describe the diagnostic pathway of tick-borne encephalitis virus infection in two horses using established molecular diagnostic methods and a novel in situ hybridization technique to differentiate between regionally important/emerging diseases for central Europe...
Jiang Z, Haughan J, Moss KL, Stefanovski D, Ortved KF, Robinson MA.Gene therapy is currently prohibited in human and equine athletes and novel analytical methods are needed for its detection. Most in vivo products use non-integrating, recombinant viral vectors derived from adeno-associated virus (AAV) to deliver transgenes into cells, where they are transcribed and translated into functional proteins. Although the majority of wild-type AAV (WTAAV) DNA is removed from recombinant AAV (rAAV) vectors, some sequences are conserved. The goal of this study was to develop a quantitative polymerase chain reaction (QPCR) screening test targeting conserved AAV sequence...
Dunnett M, Harris RC.The combined solid-phase extraction (Isolute PRS columns) and reversed-phase gradient HPLC method presented provides a sensitive, reproducible and selective quantification of carnosine, balenine, homocarnosine, histidine, 1-methylhistidine and 3-methylhistidine in equine and camel muscle and individual muscle fibres. Recoveries were 91-115%. Lower limits of detection were 0.005-0.010 mmol kg-1 dry muscle. The compounds were isolated from other physiological amino acids and small peptides and resolved within a single chromatographic run of 55 min. Concentrations of these compounds in equine myo...
Alhassan A, Govind Y, Tam NT, Thekisoe OM, Yokoyama N, Inoue N, Igarashi I.The sensitivity of LAMP, PCR and in vitro culture methods for the detection of Theileria equi and Babesia caballi was evaluated using tenfold serially diluted culture parasites. On day 1 post-culture, both T. equi and B. caballi parasites could only be observed at 1% parasite dilution from the in vitro culture method, whereas LAMP could detect up to 1 x 10(-3)% of both T. equi and B. caballi parasite dilutions, whilst PCR could detect 1 x 10(-3)% T. equi and 1 x 10(-1)% B. caballi parasite dilutions. On day 7 post-culture, the detection limit for T. equi and B. caballi in the in vitro culture ...
Deschene K, Céleste C, Boerboom D, Theoret CL.Exuberant granulation tissue (EGT), a fibrotic healing disorder resembling the human keloid, occurs almost exclusively in limb wounds of horses and may be caused in part by a relative state of hypoxia within the wound. Objective: The objectives of this study were therefore to (1) assess the effects of hypoxia on equine dermal fibroblast (EDF) proliferation and apoptosis, (2) study the effects of hypoxia on the expression of key extracellular matrix (ECM) associated proteins and determine if such effects are dependent on hypoxia-inducible factor (HIF), and (3) determine if EDFs from the body or...
Malisauskas M, Zamotin V, Jass J, Noppe W, Dobson CM, Morozova-Roche LA.The calcium-binding equine lysozyme has been found to undergo conversion into amyloid fibrils during incubation in solution at acidic pH. At pH 4.5 and 57 degrees C, where equine lysozyme forms a partially unfolded molten globule state, the protein forms protofilaments with a width of ca. 2 nm. In the absence of Ca(2+) the protofilaments are present as annular structures with a diameter of 40-50 nm. In the presence of 10 mM CaCl(2) the protofilaments of equine lysozyme are straight or curved; they can assemble into thicker threads, but they do not appear to undergo circularisation. At pH 2.0, ...
Lin Q, Rikihisa Y.Diverse p44 alleles at the p44 expression locus (p44Es) encoding surface-exposed major membrane proteins, P44s, of Anaplasma phagocytophilum were hypothesized to be garnered by recombination to enact antigenic variation. However, this hypothesis has not been proven so far, due to inability to clone this obligate intragranulocytic rickettsia. To define the p44E recombination, we developed a novel method to clone A. phagocytophilum. This isogenic cloned population containing a defined p44E was used to infect a naive horse and severe combined immunodeficiency (SCID) mice. During a 58-day infectio...
Zembowicz A, Knoepp SM, Bei T, Stergiopoulos S, Eng C, Mihm MC, Stratakis CA.Pigmented epithelioid melanocytoma (PEM) is a recently described entity comprising most cases previously described as "animal-type melanoma" and epithelioid blue nevus (EBN) occurring in patients with the multiple neoplasia syndrome Carney complex (CNC). Mutations of the protein kinase A regulatory subunit type 1alpha (R1alpha) (coded by the PRKAR1A gene) are found in more than half of CNC patients. In this study, we investigated whether PEM and EBN are related at the molecular level, and whether changes in the PRKAR1A gene status and the expression of the R1alpha protein may be involved in th...
McClain AK, McCarrel TM.Platelet-rich plasma (PRP) is a therapeutic biologic that is used for treatment of musculoskeletal pathologies in equine athletes. Due to the expense of PRP kits, and the volumes obtained, freezing aliquots for future dosing is common. Aliquots of PRP are also commonly frozen for later analysis of growth factor concentrations in in vitro research. A variety of freezing methods are used and storage duration until analysis is often not reported. The optimal frozen storage conditions and duration to maintain concentrations of commonly measured growth factors and enzymes in PRP are unknown. Our ob...
Merchant KA, Thompson DE, Xu QH, Williams RB, Loring RF, Fayer MD.Two-dimensional (2D) infrared vibrational echoes were performed on horse heart carbonmonoxymyoglobin (MbCO) in water over a range of temperatures. The A(1) and A(3) conformational substates of MbCO are found to have different dephasing rates with different temperature dependences. A frequency-frequency correlation function derived from molecular dynamics simulations on MbCO at 298 K is used to calculate the vibrational echo decay. The calculated decay shows substantial agreement with the experimentally measured decays. The 2D vibrational echo probes protein dynamics and provides an observable ...
Goodrich LR, Choi VW, Carbone BA, McIlwraith CW, Samulski RJ.Cell transplantation for the treatment of joint disease is an important clinical tool. Genetic modification of cells before transplantation has shown enhanced healing. Ex vivo genetic modification of joint tissue cells with various adeno-associated virus (AAV) serotypes has not been investigated. The transduction efficiencies of self-complementary AAV serotypes (1-6 and 8) were determined in joint tissue containing chondrocytes and synoviocytes isolated from equine models. When comparing scAAV serotypes for efficient transduction ex vivo, in chondrocytes versus synoviocytes, serotypes 6 and 2,...
Wallner B, Piumi F, Brem G, Müller M, Achmann R.Y chromosome polymorphisms such as microsatellites or single nucleotide polymorphisms represent a paternal counterpart to mitochondrial DNA (mtDNA) for evolutionary and phylogeographic studies. The use of Y chromosome haplotyping in natural populations of species other than humans is still hindered by the lack of sequence information necessary for polymorphism screening. Here we used representational difference analysis (RDA) followed by a screen of a bacterial artificial chromosome (BAC) library for repetitive sequences to obtain polymorphic Y-chromosomal markers. The procedure was performed ...
Raudsepp T, Santani A, Wallner B, Kata SR, Ren C, Zhang HB, Womack JE, Skow LC, Chowdhary BP.We herein report a detailed physical map of the horse Y chromosome. The euchromatic region of the chromosome comprises approximately 15 megabases (Mb) of the total 45- to 50-Mb size and lies in the distal one-third of the long arm, where the pseudoautosomal region (PAR) is located terminally. The rest of the chromosome is predominantly heterochromatic. Because of the unusual organization of the chromosome (common to all mammalian Y chromosomes), a number of approaches were used to crossvalidate the results. Analysis of the 5,000-rad horse x hamster radiation hybrid panel produced a map spannin...
Allen G, Yeargan M, Costa LR, Cross R.An experimental system that permits sensitive and reproducible detection of equine herpesvirus 1 (EHV-1)-specific cytotoxic T-lymphocyte (CTL) activity in the horse was developed. Peripheral blood mononuclear cells (PBMC) collected from immune horses were restimulated in vitro by culture with live EHV-1. Cytotoxic activity against virus-infected, pokeweed mitogen-stimulated lymphoblast targets was assessed in a 4-h 51Cr release assay. The optimal conditions for in vitro stimulation of equine memory CTLs and for preparation of EHV-1-infected target cells expressing viral antigens were systemati...
Klein C, Troedsson MH.Establishment and maintenance of pregnancy are critically dependent on embryo-maternal communication during the preimplantation period. The horse is one of the few domestic species in which the conceptus-derived pregnancy recognition signal has not been identified. To gain new insights into the factors released by the equine conceptus, transcriptional profiling analyses of conceptuses retrieved 8, 10, 12, and 14 days after ovulation were performed using a whole-genome microarray. Selected array data were confirmed using quantitative PCR, and the expression of proteins of interest was confirmed...
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...
Raudsepp T, Gustafson-Seabury A, Durkin K, Wagner ML, Goh G, Seabury CM, Brinkmeyer-Langford C, Lee EJ, Agarwala R, Stallknecht-Rice E, Schäffer AA....A comprehensive second-generation whole genome radiation hybrid (RH II), cytogenetic and comparative map of the horse genome (2n = 64) has been developed using the 5000rad horse x hamster radiation hybrid panel and fluorescence in situ hybridization (FISH). The map contains 4,103 markers (3,816 RH; 1,144 FISH) assigned to all 31 pairs of autosomes and the X chromosome. The RH maps of individual chromosomes are anchored and oriented using 857 cytogenetic markers. The overall resolution of the map is one marker per 775 kilobase pairs (kb), which represents a more than five-fold improvement over ...
Janssen IJ, Krücken J, Demeler J, von Samson-Himmelstjerna G.P-glycoproteins (Pgps) are suspected to mediate drug extrusion in nematodes contributing to macrocyclic lactone resistance. This association was recently shown for Parascaris Pgp-11. Ivermectin resistance was correlated with the presence of three pgp-11 single nucleotide polymorphisms and/or increased pgp-11 mRNA levels. In the present study, the ability of Pgp-11 to modulate ivermectin susceptibility was investigated by its expression in a pgp-11-deficient Caenorhabditis elegans strain. Expression of Parascaris pgp-11 in two transgenic lines significantly decreased ivermectin susceptibility i...
Creutz C, Sutin N.The kinetics of the reduction of horseheart ferricytochrome c by sodium dithionite (phosphate buffer-sodium chloride; pH 6.5, mu = 1.0, 25 degrees ) features two reaction pathways; one with the rate constant k(3) = 1.17 x 10(4) M(-1) sec(-1), the other with the rate constant k(1)k(2)/k(-1) = 6.0 x 10(4) M(-1) sec(-1). These pathways are interpreted in terms of remote attack (possibly by way of the exposed edge of the porphyrin system) and adjacent attack (requiring the opening of the heme crevice). The limiting rate for the adjacent pathway (k(1) = 30 sec(-1)) is in good agreement with the rat...
Al Jassim RA, Scott PT, Trebbin AL, Trott D, Pollitt CC.Seventy-two lactic acid producing bacterial isolates (excluding streptococci) were cultured from the gastrointestinal tract of six horses. Two of the horses were orally dosed with raftilose to induce lactic acidosis and laminitis while the remaining four were maintained on a roughage diet. Near complete 16S rDNA was amplified by PCR from the genomic DNA of each isolate. Following RFLP analysis with the restriction enzymes MboI, HhaI and HinfI, the PCR products from the 18 isolates that produced L- and/or D-lactate were subsequently cloned and sequenced. DNA sequence analysis indicated that the...
Frampton AR, Uchida H, von Einem J, Goins WF, Grandi P, Cohen JB, Osterrieder N, Glorioso JC.To initiate infection, equine herpesvirus type 1 (EHV-1) attaches to heparan sulfate on cell surfaces and then interacts with a putative glycoprotein D receptor(s). After attachment, virus entry occurs either by direct fusion of the virus envelope with the plasma membrane or via endocytosis followed by fusion between the virus envelope and an endosomal membrane. Upon fusion, de-enveloped virus particles are deposited into the cytoplasm and travel to the nucleus for viral replication. In this report, we examined the mechanism of EHV-1 intracellular trafficking and investigated the ability of EH...
Nasir L, Reid SW.The sarcoid is a benign locally invasive dermal fibroblastic lesion, commonly affecting horses and donkeys. The aetiology of the equine sarcoid is equivocal. Bovine papillomaviral (BPV) DNA (type 1/2) is frequently demonstrable in equine sarcoid tumour biopsies. However, the exact role of the virus in the disease process and its contribution to the phenotypic differences in sarcoids is not known. It was sought to assess the transcriptional activity of BPV-1 found in sarcoid tissues. Of 20 tumours examined, 18 were positive for E2 expression and ten positive for L1 expression. Viral oncogenes E...
Hooper PT, Ketterer PJ, Hyatt AD, Russell GM.Laboratory examinations of equine morbillivirus included experimental reproductions of the disease caused by the virus by transmission of mixed lung and spleen taken from two field equine cases into two horses and by inoculating tissue culture virus into a further two horses. The most distinctive gross lesions of the diseases that developed in three of the horses was that of pulmonary edema characterized by gelatinous distension of subpleural lymphatics. Histologically, the lesions in the lungs were those of serofibrinous alveolar edema, alveolar macrophages, hemorrhage, thrombosis of capillar...
Nergadze SG, Lupotto M, Pellanda P, Santagostino M, Vitelli V, Giulotto E.The insertion of mitochondrial DNA in the nuclear genome generates numts, nuclear sequences of mitochondrial origin. In the horse reference genome, we identified 82 numts and showed that the entire horse mitochondrial DNA is represented as numts without gross bias. Numts were inserted in the horse nuclear genome at random sites and were probably generated during the repair of DNA double-strand breaks. We then analysed 12 numt loci in 20 unrelated horses and found that null alleles, lacking the mitochondrial DNA insertion, were present at six of these loci. At some loci, the null allele is prev...
González-Fernández L, Ortega-Ferrusola C, Macias-Garcia B, Salido GM, Peña FJ, Tapia JA.Protein tyrosine kinases have important roles in spermatozoa; however, little is known about the presence and regulation in these cells of their counterparts in signaling, namely, protein tyrosine phosphatases (PTPs) and dual-specificity phosphatases (DSPs). The objectives of the present study were to identify PTPs and DSPs in boar, stallion, and dog spermatozoa; to characterize their subcellular distribution; and to investigate the roles of tyrosine phosphatases in maintenance of protein tyrosine phosphorylation level and in sperm motility. Using Western blotting with specific antibodies in b...
Luo J, Bruice TC.Molecular dynamics and cross-correlation analysis of the horse liver alcohol dehydrogenase HLADH.NAD(+).PhCH(2)O(-) complex has established anticorrelated motions between the NAD(+)-binding domain and other portions of the enzyme. Four pairs of anticorrelated interactions are (i and ii) cofactor-binding domain: C(alpha) of V292 and the CG1 of V203 with C7 of PhCH(2)O(-); (iii) cofactor-binding domain: amide carbonyl oxygen of I318 with amide N of H67; and (iv) cofactor domain: C(alpha) of T178 with carbonyl oxygen of L141. The average distances between pairs are 9.2 A for i, 8.2 A for ii, 14.7...
Woolcock JB, Farmer AM, Mutimer MD.The development of a selective medium for the isolation of Corynebacterium equi is described. The medium has been used to examine fecal samples from 127 horses of which 90 have been found to carry the organism.
Zhang X, Li C, Nan Z.An evaluation was performed on the influence of mowing height (2.5, 5.0 or 7.5 cm) and mowing frequency (weekly or fortnightly) on levels of ergot alkaloids (ergine and ergonovine) formed in drunken horse grass, Achnatherum inebrians, grown under greenhouse conditions. Samples were taken monthly and alkaloids were extracted and analyzed by reverse-phase HPLC. Alkaloid levels increased more or less linearly with plant age over the first four months following establishment. Levels were higher in samples cut fortnightly compared to those cut weekly, and were higher when plants were cut at a mowin...
Tallmadge RL, Lear TL, Antczak DF.The availability of a contig of bacterial artificial chromosome (BAC) clones spanning the equine major histocompatibility complex (MHC) made possible a detailed analysis of horse MHC class I genes. Prior to this study, only a single horse MHC class I gene had been sequenced at the genomic level. Although many ( approximately 60) MHC class I cDNA sequences had been determined and published, from this information, it was not possible to determine how many class I loci are expressed in horses or to assign individual sequences to allelic series. In this study, 15 MHC class I genes were identified ...
Rwambo PM, Issel CJ, Hussain KA, Montelaro RC.A neutralization escape mutant (A/1 E) of equine infectious anemia virus was isolated after 13 passages in cell culture in the presence of serum containing antibodies to type- and group-specific determinants of EIAV envelope glycoproteins. Loss of neutralization by the selecting serum correlated with loss of two epitopes in the major envelope glycoprotein gp90 of A/1 E which were present in a parallel variant isolated from a persistently infected pony.
