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.
Lear TL, Bailey E.Cytogenetic analyses of horses have benefited the horse industry by identifying chromosomal aberrations causing congenital abnormalities, embryonic loss and infertility. Technical advances in cytogenetics enabled the identification of chromosome specific aberrations. More recently, advances in genomic tools have been used to more precisely define chromosome abnormalities. In this report we review the history of equine clinical cytogenetics, identify historical landmarks for equine clinical cytogenetics, discuss how the current use of genomic tools has benefited this area, and how future genomi...
Quan M, van Vuuren M, Howell PG, Groenewald D, Guthrie AJ.Between 2004 and 2006, 145 African horse sickness viruses (AHSV) were isolated from blood and organ samples submitted from South Africa to the Faculty of Veterinary Science, University of Pretoria. All nine serotypes were represented, with a range of 3-60 isolates per serotype. The RNA small segment 10 (S10) nucleotide sequences of these isolates were determined and the phylogeny investigated. AHSV, bluetongue virus (BTV) and equine encephalosis virus (EEV) all formed monophyletic groups and BTV was genetically closer to AHSV than EEV. This study confirmed the presence of three distinct S10 ph...
Brandt S, Haralambus R, Shafti-Keramat S, Steinborn R, Stanek C, Kirnbauer R.Bovine papillomavirus type 1 or 2 (BPV-1, BPV-2) are accepted causal factors in equine sarcoid pathogenesis. Whereas viral genomes are consistently found and expressed within lesions, intact virions have never been detected, thus permissiveness of sarcoids for BPV-1 replication remains unclear. To reassess this issue, an immunocapture PCR (IC/PCR) was established using L1-specific antibodies to capture L1-DNA complexes followed by amplification of the viral genome. Following validation of the assay, 13 sarcoid-bearing horses were evaluated by IC/PCR. Samples were derived from 21 tumours, 4 per...
Vonaparti A, Lyris E, Panderi I, Koupparis M, Georgakopoulos C.Two simple and rapid LC/MS methods with direct injection analysis were developed and validated for the quantification and identification of hydrocortisone in equine urine using the same sample preparation but different mass spectrometric systems: ion trap mass spectrometry (IT-MS) and time-of-flight mass spectrometry (TOF-MS). The main advantage of the proposed methodology is the minimal sample preparation procedure, as particle-free diluted urine samples were directly injected into both LC/MS systems. Desonide was used as internal standard (IS). The linear range was 0.25-2.5 microg ml(-1) for...
Westermann CM, Dorland L, Votion DM, de Sain-van der Velden MG, Wijnberg ID, Wanders RJ, Spliet WG, Testerink N, Berger R, Ruiter JP, van der Kolk JH.The aim of the current study was to assess lipid metabolism in horses with atypical myopathy. Urine samples from 10 cases were subjected to analysis of organic acids, glycine conjugates, and acylcarnitines revealing increased mean excretion of lactic acid, ethylmalonic acid, 2-methylsuccinic acid, butyrylglycine, (iso)valerylglycine, hexanoylglycine, free carnitine, C2-, C3-, C4-, C5-, C6-, C8-, C8:1-, C10:1-, and C10:2-carnitine as compared with 15 control horses (12 healthy and three with acute myopathy due to other causes). Analysis of plasma revealed similar results for these predominantly...
Endo A, Roos S, Satoh E, Morita H, Okada S.Two strains of lactic acid bacteria were isolated from faeces of two actively racing thoroughbred horses. The isolates formed a subcluster in the Lactobacillus reuteri phylogenetic group, closely related to Lactobacillus fermentum, L. gastricus, L. ingluviei and L. mucosae, by phylogenetic analysis based on 16S rRNA gene sequences. Levels of DNA-DNA relatedness revealed that the isolates belonged to the same taxon and were genetically separated from their phylogenetic relatives. Biochemical and physiological characteristics also distinguished the isolates from their phylogenetic relatives. The...
Hastie PM, Mitchell K, Murray JA.There is a need to further our understanding of the role that the equine hindgut ecosystem plays in digestive processes and diseases. The aim of the present study was to utilise the real-time PCR technique to determine the abundance of candidate cellulolytic (Ruminococcus flavefaciens; Fibrobacter succinogenes) and non-cellulolytic (Streptococcus bovis) bacteria in lumen contents from the caecum, ventral and dorsal colon, and rectum of healthy horses (n 14). Total DNA was extracted from frozen and lyophilised lumen contents, and PCR primers and Taqman probes were designed based on 16S rDNA seq...
Giordano A, Rossi G, Pieralisi C, Paltrinieri S.The ADVIA 120 is an automated laser cell counter widely used in veterinary medicine. Although specific software for equine samples is available and validated, only a few reports have been published comparing the ADVIA 120 with other methods for equine hemogram evaluation. Objective: The purpose of this study was to compare the hematologic values and reference intervals obtained on the ADVIA 120 with those obtained on an impedance cell counter and manual differential counts in healthy horses. Methods: EDTA-anticoagulated blood samples were obtained from 114 clinically healthy horses of various ...
Owens SD, Snipes J, Magdesian KG, Christopher MM.Blood typing before transfusion minimizes the risk of transfusion reactions and prevents immunization of the recipient against incompatible RBC antigens. The major RBC antigens that warrant identification before packed RBC or whole blood transfusions in horses are Ca and Aa. Standard blood-typing protocols are time-consuming (2.5-3.0 hours) and impractical in emergency settings. Objective: The purpose of this study was to determine whether equine RBCs could be typed for Ca and Aa antigens using sera from horses with RBC antibodies in a modified rapid (15 minute) blood-typing protocol. Methods:...
Colas C, Garcia P, Popot MA, Bonnaire Y, Bouchonnet S.Solid-phase extraction cartridges among those usually used for screening in horse doping analyses are tested to optimize the extraction of harpagoside (HS), harpagide (HG), and 8-para-coumaroyl harpagide (8PCHG) from plasma and urine. Extracts are analyzed by liquid chromatography coupled with multi-step tandem mass spectrometry. The extraction process retained for plasma applies BondElut PPL cartridges and provides extraction recoveries between 91% and 93%, with RSD values between 8 and 13% at 0.5 ng/mL. Two different procedures are needed to extract analytes from urine. HS and 8PCHG are extr...
Matiasek K, Gais P, Rodenacker K, Jütting U, Tanck JJ, Schmahl W.Stereological techniques have been increasingly employed for assessment and characterization of neuromuscular diseases in humans and animals. As an adjunct to histopathology, morphometrical algorithms provide quantitative evidence of the peripheral nerve composition, thereby shedding light on its fibre characteristics and basic electrophysiological properties. In the horse, stereological investigations already have focussed on the recurrent laryngeal, deep peroneal and lateral palmar nerves (LPN). Of these, only the latter is suitable for taking biopsies in clinical settings, however, it does ...
Andrews FM, Buchanan BR, Elliott SB, Al Jassim RA, McGowan CM, Saxton AM.Volatile fatty acids, byproducts of carbohydrate fermentation by resident bacteria, have been implicated in causing nonglandular (NG) gastric ulcers. Lactic acid (LA), also produced by stomach bacteria, may cause gastric ulcers when exposed to the equine NG mucosa. Objective: To investigate the in vitro effects of LA on equine NG mucosa bioelectric properties, sodium transport and tissue resistance. Methods: Gastric tissues obtained from 13 mature horses were studied in Ussing chambers. Short-circuit current (Isc) and potential difference (PD) were measured, and electrical resistance (R) and c...
Lu Z, Branscum AJ, Shuck KM, Zhang J, Dubovi EJ, Timoney PJ, Balasuriya UB.Two previously developed TaqMan fluorogenic probe-based 1-tube real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assays (T1 and T2) were compared and validated for the detection of Equine arteritis virus (EAV) nucleic acid in equine semen and tissue culture fluid (TCF). The specificity and sensitivity of these 2 molecular-based assays were compared to traditional virus isolation (VI) in cell culture. The T1 real-time RT-PCR had a higher sensitivity (93.4%) than the T2 real-time RT-PCR (42.6%) for detection of EAV RNA in semen. However, the T1 real-time RT-PCR was les...
Yamanaka T, Tsujimura K, Kondo T, Matsumura T.In this study, we evaluated whether five rapid antigen detection kits for human influenza could be used for the diagnosis of equine influenza (EI). Limiting dilution analyses showed that Directigen Flu A+B and ESPLINE INFLUENZA A&B-N had the highest sensitivities to equine-2 influenza viruses (EIVs) among the kits investigated. From the results of virus detection in nasal swabs taken from horses infected with EIV, these two kits could produce positive results in reasonable agreement with those obtained by virus isolation or RT-PCR, suggesting that these kits could be useful for rapid diagn...
Menzies-Gow NJ, Bailey SR, Berhane Y, Brooks AC, Elliott J.To determine the effect of endotoxin (lipopolysaccharide [LPS]) on vasoactive mediator production by cultured equine digital vein endothelial cells (EDVECs). Methods: EDVECs obtained from forelimb digital veins of 7 healthy adult horses. Methods: EDVECs were incubated with or without LPS (1 microg/mL) for 0, 2, 4, 6, 22, and 24 hours. The EDVECs were incubated for 18 hours with LPS (10 pg/mL to 1 microg/mL) with or without ibuprofen, cycloheximide, or L-nitroarginine methyl ester. Medium concentrations of prostacyclin, cyclic guanosine monophosphate, endothelin-1, and thromboxane A(2) were det...
Varhac R, Antalík M.A relation between pH-induced conformational transitions of horse heart ferricytochrome c and the kinetics of external ligand coordination to heme iron was investigated by optical spectroscopy, circular dichroism and viscometry. The dependencies of both the association, k (a), and dissociation rate constants of cyanide binding on pH were determined from kinetic measurements. The association rate constant exhibits a bell-shaped form of dependence on pH in the region where this protein unfolds. The maximum of the dependence of k (a) on pH is found to be coincident with the pK values of conformat...
Royo LJ, Fernández I, Azor PJ, Alvarez I, Pérez-Pardal L, Goyache F.The aim of this note is to describe a reliable, fast, and cost-effective real-time PCR method for routine genotyping of mutations responsible for most coat color variation in horses. The melanocortin-1 receptor, Agouti-signaling peptide, and membrane-associated transporter protein alleles were simultaneously determined using 2 PCR protocols. The assay described here is an alternative method for routine genotyping of a defined number of polymorphisms. Allelic variants are detected in real time and no post-PCR manipulations are required, therefore limiting costs and possible carryover contaminat...
Iniesta J, Cooper HJ, Marshall AG, Heptinstall J, Walton DJ, Peterson IR.The iodination of proteins remains a useful tool in biochemistry for radiolabelling. However, chemical or enzymatic iodination is difficult to control and can give deleterious polyiodination. Previously, we have shown that electrooxidation with nitrite is a rapid method for the selective nitration of tyrosine residues in proteins. In principle, it should be possible to substitute a number of electrooxidisable anions into the tyrosine phenol ring. Electrochemical iodination is more difficult to control than nitration because the rapid anodic oxidation of I(-) leads to persistent formation of th...
Posthuma CC, Pedersen KW, Lu Z, Joosten RG, Roos N, Zevenhoven-Dobbe JC, Snijder EJ.The replication/transcription complex of the arterivirus equine arteritis virus (EAV) is associated with paired membranes and/or double-membrane vesicles (DMVs) that are thought to originate from the endoplasmic reticulum. Previously, coexpression of two putative transmembrane nonstructural proteins (nsp2 and nsp3) was found to suffice to induce these remarkable membrane structures, which are typical of arterivirus infection. Here, site-directed mutagenesis was used to investigate the role of nsp3 in more detail. Liberation of the hydrophobic N terminus of nsp3, which is normally achieved by c...
Wnuk M, Bugno M, Slota E.Recently, molecular techniques have become an indispensable tools for cytogenetic research. Especially, development of in situ techniques made possible detection at the chromosomal level, genes as well as repetitive sequences like telomeres or the DNA component of telomeres. One of these methods is primed in situ DNA synthesis (PRINS) using an oligonucleotide primer complementary to the specific DNA sequence. In this report we described application of PRINS technique with telomere human commercial kit to telomere sequences identification. This commercial kit may be use to visualization of inte...
Meng Q, Li S, Liu L, Xu J, Liu Y, Zhang Y, Zhang X, Shao Y.Equine Infectious Anemia Virus (EIAV), like other lentiviruses, has a transmembrane glycoprotein with an unusually long cytoplasmic tail (CT). Viral envelope (Env) proteins having CT truncations just downstream the putative membrane-spanning domain (PMSD) are assumed to exist among all wild-type budded virions, and also in some cell-adapted strains. To determine whether CT-truncated Env proteins can cause particularly deleterious effects on the Env expressing cells and/or their neighboring cells, plasmids encoding codon-optimized env gene including full-length (pE863) or CT-truncated (pE686* a...
Wernery U, Knowles NJ, Hamblin C, Wernery R, Joseph S, Kinne J, Nagy P.A virus was isolated from aborted dromedary (Camelus dromedarius) fetuses during an abortion storm in Dubai, United Arab Emirates. Laboratory investigations showed the causative agent to be indistinguishable from equine rhinitis A virus (ERAV), a picornavirus. Two pregnant dromedaries experimentally infected with the camel virus isolate both aborted and an identical virus was reisolated from both fetuses, thus confirming the diagnosis. The extremely high prevalence of antibody (>90 %) and the high titres recorded against ERAV in the dromedary herd clearly showed that ERAV does infect dromedari...
Popot MA, Woolfitt AR, Garcia P, Tabet JC.The insulin-like-growth factor (IGF-I) peptide is considered to be the main indirect marker for growth hormone administration (GH) in a horse. Further to a previous investigation on measurement of IGF-I in plasma samples by mass spectrometry, this study focuses on quantitative and qualitative analysis of intact IGF-I in horse plasma. First, protein-transposing software has been developed for IGF-I to facilitate its quantification by HPLC-electrospray-ion-trap mass spectrometry. Second, product-ion scan experiments on IGF-I have been conducted on standard samples, non-fortified equine plasma sa...
Berg LC, Lenz J, Kjelgaard-Hansen M, Thomsen PD, Jacobsen S.More sensitive and specific diagnostic methods for early detection of changes in the joint cartilage are needed. Cartilage-derived retinoic acid-sensitive protein (CD-RAP) is a potential marker of cartilage synthesis and regeneration. This is the first study on equine CD-RAP. Objective: To evaluate the ability of a commercially available human sandwich ELISA assay to detect equine CD-RAP in synovial fluid from healthy and diseased joints. Methods: Synovial fluid was collected from 28 horses with no signs of joint disease and from 5 with induced inflammatory arthritis. CD-RAP concentrations wer...
Duthie S, Mills H, Burr P.Infection with equine arteritis virus is a notifiable disease with sporadic occurrence in the UK. As stallions may harbour the virus after infection, horses are screened for exposure by serological testing prior to breeding. The virus neutralisation test is considered the 'gold standard' serological screening test, but it is time-consuming and labour intensive; consequently there is a move towards more rapid screening methodology. In this study, a commercially available EVA antibody ELISA is assessed. The ELISA performed poorly with a specificity [corrected] of 26% and a sensitivity [corrected...
Matsumura Y, Li J, Ikeguchi M, Kihara H.Acidic buffer conditions are known to stabilize helix-rich states of even those proteins with a predominantly beta-sheet native secondary structure. Here we investigated whether such states also exist under alkaline buffer conditions. The guanidine hydrochloride (GuHCl)-induced unfolding transition and kinetic refolding of equine beta-lactoglobulin (ELG) by GuHCl-jump were investigated at pH 8.7 by far-ultraviolet circular dichroism. We found that an equilibrium intermediate appeared in 45% ethylene glycol (EGOH) buffer with 1.5 M GuHCl. The intermediate is rich in non-native alpha-helix, whic...
Delesalle C, van Acker N, Claes P, Deprez P, de Smet I, Dewulf J, Lefebvre RA.Prokinetic drugs used to treat gastrointestinal ileus in man have equivocal results in horses. In man, prokinetic drugs have 5-hydroxytryptamine4(5-HT4) receptors as their target, but little is known about the 5-HT-receptor subtypes in the equine small intestine. Objective: Functional and immunohistochemical identification of the serotonin receptor subtype(s) responsible for the 5-HT induced contractile response in the equine circular jejunum. Methods: Isometric organ-bath recordings were carried out to assess spontaneous and drug-evoked contractile activity of equine circular jejunum. Histolo...
Kwok WH, Leung DK, Leung GN, Tang FP, Wan TS, Wong CH, Wong JK.In September 2005, our laboratory detected the presence of 4-androstene-3,17-dione and androsterone in a standard steroid screen of a post-race gelding urine sample received from an overseas authority. All other urine samples from the same batch tested negative. Subsequent gas chromatography/mass spectrometry (GC/MS) confirmatory analyses, however, repeatedly failed to detect any amount of 4-androstene-3,17-dione and androsterone in the suspicious sample. On the other hand, identical results were obtained when the initial GC/MS screening method was repeated on the suspicious sample as well as ...
Lawler JB, Hassel DM, Magnuson RJ, Hill AE, McCue PM, Traub-Dargatz JL.To determine the adsorptive capability of di-tri-octahedral smectite (DTOS) on Clostridium perfringens alpha, beta, and beta-2 exotoxins and equine colostral antibodies. Methods: 3 C perfringens exotoxins and 9 colostral samples. Methods: Alpha, beta, and beta-2 exotoxins were individually co-incubated with serial dilutions of DTOS or bismuth subsalicylate, and the amount of toxin remaining after incubation was determined via toxin-specific ELISAs. Colostral samples from healthy mares were individually co-incubated with serial dilutions of DTOS, and colostral IgG concentrations were determined...
Welsch U, Schumacher U, Buchheim W, Schinko I, Jenness P, Patton S.A specific secretory product of the lactating mammary gland are triglyceride fat globules which are enveloped by a very complex membrane, the milk fat globule membrane (MFGM). In different mammalian species (man, rhesus monkey, horse, goat, sheep, cow, grey seal, camel, alpaka) the glycoproteins of this membrane have been analyzed by gel electrophoresis, Western blotting and lectin histochemistry. A remarkable intra- and interspecific variability of these glycoproteins has been detected pointing to so far unknown physiological adaptions, which may play a role in the intestine of the new born. ...
Norris JK, Slusarewicz P, Nielsen MK.The efficacy of anthelmintic treatments against populations of endoparasites infecting livestock throughout the world is decreasing. To mitigate this, the use of fecal egg counts is recommended to determine both the necessity, and to ensure the appropriate choice, of anthelmintic treatment. Traditionally, and in order to facilitate easier identification and/or enumeration, samples are analysed after separating eggs from other fecal particulates by exposing them to a solution with a density higher than that of the eggs, but lower than the remaining fecal contents. While many parasite egg flotat...
Zientara S, Sailleau C, Moulay S, Wade-Evans A, Cruciere C.The development of a coupled reverse transcriptase-polymerase chain reaction assay (RT-PCR) is described for the detection of African horse sickness virus (AHSV) double-stranded RNA. Genome segments 7 and 10 were chosen as target templates for primers selected for use in the RT-PCR. Using these AHSV-specific primers all 9 serotypes were detectable. The sensitivity and specificity of the RT-PCR results were compared to those obtained by competition ELISA.
Teixeira-Soares CM, Viana AGA, Ribeiro IM, Silva KDP, Sancler-Silva YFR, Machado-Neves M.Endometritis is a relevant cause of subfertility in mares. However, the accurate diagnosis, essential for effective treatment, can be difficult due to the variability of results and interpretations resulting from different examination methods and sample collection techniques. The present work compared gynecological evaluation methods and sample collection techniques to diagnose endometritis in subfertile mares. Forty animals with a history of subfertility were selected for gynecological evaluation using clinical methodologies, such as perineal conformation, transrectal palpation and ultrasonog...
Theurillat R, Knobloch M, Levionnois O, Larenza P, Mevissen M, Thormann W.A robust CE method for the simultaneous determination of the enantiomers of ketamine and norketamine in equine plasma is described. It is based upon liquid-liquid extraction of ketamine and norketamine at alkaline pH from 1 mL plasma followed by analysis of the reconstituted extract by CE in the presence of a pH 2.5 Tris-phosphate buffer containing 10 mg/mL highly sulfated beta-CD as chiral selector. Enantiomer plasma levels between 0.04 and 2.5 microg/mL are shown to provide linear calibration graphs. Intraday and interday precisions evaluated from peak area ratios (n = 5) at the lowest calib...
Yilma T, McGuire TC, Perryman LE.Equine dermal cells induced with poly I:C + DEAE-dextran produced low levels of interferon tentatively classified as equine interferon beta (EqIFN-beta). In contrast, dermal cells initially primed with EqIFN-beta and then superinduced with poly I:C + DEAE-dextran in the presence of cycloheximide and actinomycin D produced greater than 100-fold EqIFN-beta. Equine blood mononuclear cells induced with Newcastle disease virus and phytohemagglutinin produced high levels of interferons tentatively classified as equine interferon alpha (EqIFN-alpha) and equine interferon gamma (EqIFN-gamma), respecti...
Vachon AM, McIlwraith CW, Keeley FW.Periosteal autografts were used for repair of large osteochondral defects in 10 horses aged 2 to 3 years old. In each horse, osteochondral defects measuring 1.0 x 1.0 cm2 were induced bilaterally on the distal articular surface of each radial carpal bone. Control and experimental defects were drilled. Periosteum was harvested from the proximal portion of the tibia and was glued into the principal defects, using a fibrin adhesive. Control defects were glued, but were not grafted. Sixteen weeks after the grafting procedure, the quality of the repair tissue of control and grafted defects was asse...
Beretta C, Fadini L, Stracciari JM, Montesissa C.Febantel and one of its main metabolites, febantel sulphoxide, are chemically modified to only a slight extent when incubated in vitro with sheep and cattle ruminal fluids; other major metabolites, fenbendazole and oxfendazole, are respectively, oxidized to oxfendazole and reduced to fenbendazole. Febantel is negligibly metabolized by hepatic cytosol fractions but microsome preparations effect more extensive metabolic transformations. Important differences in this respect were found between microsome preparations from rat, horse, pig, cattle, sheep, chicken and trout livers.
Okada A, Suganuma S, Badr Y, Omatsu T, Mizutani T, Ohya K, Fukushi H.VP22 is a major tegument protein of equine herpesvirus type 1 (EHV-1). In the present study, we examined functions of VP22 in EHV-1 replication by viral protein expression analyses in cells infected with the VP22-deficient virus. The expressions of several viral proteins in the cells infected with the VP22-deficient virus were lower than those in the cells infected with the parent virus. One of the weakly expressed proteins was identified as ICP4, which is a major regulatory protein encoded by an immediate early gene of EHV-1. A real-time PCR analysis showed that the mRNA expression of ICP4 wa...
Leidinger EF, Leidinger J, Figl J, Rumpler B, Schwendenwein I.Despite the increasing popularity of Icelandic horses, published reference intervals (RIs) in this breed are rare. Due to their isolation and their small gene pool, alterations in some variables are likely and some possible breed-specific peculiarities have been described. The purpose of the present study was the establishment of comprehensive RIs in Icelandic horses according to recently published guidelines. In a prospective observational study, blood samples were collected from the jugular vein of 142 Icelandic horses into EDTA and serum tubes. Reference intervals were established for haema...
Sofronic-Milosavljevic L, Pozio E, Patrascu IV, Skerovic N, Gomez Morales MA, Gamble HR.From 1998 to 2000, 5,267 horse sera were collected from several Trichinella regions in Romania. Sera were initially screened in laboratories in Romania, Serbia and Italy with an ELISA and a Western blot (Wb) using an excretory/secretory (ES) antigen and several conjugates (protein A, protein G, and sheep or goat anti-horse). Differences in serology results were obtained among the different conjugates and also between ELISA and Wb. Depending on the test used, specific antibodies were found at a prevalence rate of 3-6% of horses. Serum samples classified as positive were tested again by ELISA us...
Gregory CR, Latimer KS, Niagro FD, Campagnoli RP, Steffens WL, Ritchie BW.Eastern equine encephalomyelitis (F.EE) virus was detected in infected formalin-fixed horse and emu tissues and in infected chicken embryo fibroblasts. Results of in situ hybridization using a digoxigenin-labeled 40-base DNA probe complementary to a conserved region of the EEE virus RNA compared favorably with results of both virus isolation and serum neutralization tests. This technique may be useful for diagnosis of EEE virus infection in various animal species, especially when fresh tissues are not available for analysis, and also will provide a means for studying the involvement of alphavi...
Choi TLS, Kwok KY, Kwok WH, Tsoi YYK, Wong JKY, Wan TSM.Anabolic and androgenic steroids (AAS) are banned substances in both human and equine sports. They are often administered intramuscularly to horses in esterified forms for the purpose of extending their time of action. The authors' laboratory has previously reported an UHPLC/HRMS method using quadrupole-Orbitrap mass spectrometer in full scan and parallel reaction monitoring (PRM) mode for the detection of 48 AAS and/or their esters in horse hair. However, two injections were required due to the long duty cycle time. In this paper, an UHPLC/HRMS method using multiplexed targeted MS mode was de...
Koepf EK, Hewitt J, Vo H, Macgillivray RT, Burtnick LD.We have generated and characterized the cDNA from equine smooth muscle that encodes gelsolin, an actin-modulating protein. Overlapping cDNA clones synthesized by the reverse transcriptase/polymerase chain reaction and clones isolated from a horse genomic library provided the complete primary structure for the intracellular isoform of gelsolin, while cDNA complemented with protein sequence data produced the full-length primary transcript of the gelsolin isoform found circulating in equine plasma. The deduced amino acid sequences of the intracellular and secreted versions of equine gelsolin infe...
Colas C, Garcia P, Popot MA, Bonnaire Y, Bouchonnet S.Solid-phase extraction cartridges among those usually used for screening in horse doping analyses are tested to optimize the extraction of harpagoside (HS), harpagide (HG), and 8-para-coumaroyl harpagide (8PCHG) from plasma and urine. Extracts are analyzed by liquid chromatography coupled with multi-step tandem mass spectrometry. The extraction process retained for plasma applies BondElut PPL cartridges and provides extraction recoveries between 91% and 93%, with RSD values between 8 and 13% at 0.5 ng/mL. Two different procedures are needed to extract analytes from urine. HS and 8PCHG are extr...
Rubessa M, Feugang JM, Kandel ME, Schreiber S, Hessee J, Salerno F, Meyers S, Chu I, Popescu G, Wheeler MB.The capacity for microscopic evaluation of sperm is useful for assisted reproductive technologies (ART), because this can allow for specific selection of sperm cells for in vitro fertilization (IVF). The objective of this study was to analyze the same sperm samples using two high-resolution methods: spatial light interference microscopy (SLIM) and atomic force microscopy (AFM) to determine if with one method there was more timely and different information obtained than the other. To address this objective, there was evaluation of sperm populations from boars and stallions. To the best of our k...
Bello TR, Allen TM.To compare the McMaster and centrifugal flotation techniques and larval culture for recovery of cyathostomin (small strongyle) eggs from the feces of horses. Methods: Fecal samples from 101 horses. Methods: In experiment I, homogenized fresh feces from a single horse were randomly subsampled by use of each technique for 10 replicates. In experiment II, samples from 43 horses that had no anthelmintic treatment were analyzed by use of McMaster, centrifugal flotation, and larval culture techniques. In experiment III, 57 horses were treated with an anthelmintic by owners, and fecal samples were an...
Reuter G, Pfeil R, Kamerling JP, Vliegenthart JF, Schauer R.The novel sialic acid 4-O-acetyl-9-O-lactyl-N-acetylneuraminic acid has been identified as a constituent of horse submandibular gland glycoproteins in addition to the already known equine sialic acids, N-acetylneuraminic acid, 4-O-acetyl-N-acetylneuraminic acid, 9-O-acetyl-N-acetylneuraminic acid, 4,9-di-O-acetyl-N-acetylneuraminic acid, N-glycolylneuraminic acid, 4-O-acetyl-N-glycolylneuraminic acid and 9-O-acetyl-N-glycolylneuraminic acid. The structure has been established by combined gas-liquid chromatography-mass spectrometry.
Battaglia F, Meucci V, Tognetti R, Bonelli F, Sgorbini M, Lubas G, Pretti C, Intorre L.In human medicine, procalcitonin (PCT), the precursor of calcitonin, is used for the rapid identification of the origin and severity of sepsis. In veterinary medicine, PCT has been studied in horses, cattle, and dogs, but the use of PCT in diagnostic and/or prognostic settings is not possible because of the lack of validated assays to obtain reference ranges. The aim of the present study was the investigation of commercially available ELISA kits for the detection of canine and equine PCT in plasma samples. Validation of the ELISA kits was performed by using species-specific recombinant protein...
D'Silva C.Equine liver glutathione S-transferase has been shown to consist of two identical subunits of apparent Mr 25,500 and a pl of 8.9. Kinetic data at pH 6.5 with 1-chloro-2,4-dinitrobenzene as a substrate suggests a random rapid-equilibrium mechanism, which is supported by inhibition studies using glutathione analogues. S-(p-Bromobenzyl)glutathione and the corresponding N alpha-, CGlu- and CGly-substituted derivatives have been found, at pH 6.5, to be linear competitive inhibitors, with respect to GSH, of glutathione transferase. N-Acetylation of S-(p-bromobenzyl)glutathione decreases binding by 1...
von Engelhardt W, Burmester M, Hansen K, Becker G.Unidirectional fluxes of short-chain fatty acids across pig, sheep and pony caecum, proximal and distal colon were studied under short-circuit current conditions in Ussing chambers. Findings are compared with results from guinea pig. Marked species differences are apparent; highest mucosal-to-serosal fluxes of acetate, propionate and butyrate were seen in guinea pig, lower values in pig and smallest fluxes in sheep and pony. Segmental differences between caecum, proximal and distal colon exist mainly in guinea pig and are less developed in pig, sheep and pony. Inhibition of Na+/H+ exchange by ...
Calvete JJ, Reinert M, Sanz L, Töpfer-Petersen E.Boar and stallion seminal plasmas were fractionated using affinity chromatography on heparin-Sepharose. In both species, among other proteins, the heparin-binding (H+) and non-heparin-binding (H-) fractions each contained glycoforms of either porcine PSP-I or equine HSP-1 and HSP-2. However, porcine H+/PSP-I eluted as a monomeric protein, whereas H-/PSP-I formed a heterodimer with PSP-II, another major seminal plasma protein. On the other hand, the stallion proteins H+/HSP-1 and H+/HSP-2 eluted together as an aggregate of relative molecular mass (M(r)) 90,000, whereas H-/HSP-1 and H-/HSP-2 elu...
Turner RM, Zeng W.Studies in laboratory rodents are shedding light on the pathophysiology of testicular ageing and now suggest a complicated basis for age-related declines in testicular function. A highly significant contributor to infertility may involve failure of specific and complex testicular microenvironments (niches) comprised of a variety of cellular and molecular components. Our laboratory has applied testis tissue xenografting to the study of testicular ageing in the stallion. Using this technique, we have confirmed that the disease is tissue autologous. As would be expected from a tissue autologous d...
Holl HM, Gao S, Fei Z, Andrews C, Brooks SA.Laminitis, the structural failure of interdigitated tissue that suspends the distal skeleton within the hoof capsule, is a devastating disease that is the second leading cause of both lameness and euthanasia in the horse. Current transcriptomic research focuses on the expression of known genes. However, as this tissue is quite unique and equine gene annotation is largely derived from computational predictions, there are likely yet uncharacterized transcripts that may be involved in the etiology of laminitis. In order to create a novel annotation resource, we performed whole transcriptome seque...
Park DH, Plapp BV.The E and S isoenzymes of horse liver alcohol dehydrogenase differ by 10 amino acid residues, but only the S isoenzyme is active on 3 beta-hydroxysteroids. This functional difference was correlated to the differences in structures of the isoenzymes by characterizing a series of chimeric enzymes, which could represent intermediates in the evolution of catalytic activity. Deletion of Asp-115 from the E isoenzyme created the E/D115 delta enzyme that is active on steroids. The deletion alters the substrate binding pocket by moving Leu-116, which sterically hinders binding of steroids in the E isoe...
Elroy NN, Rogers J, Mafi GG, VanOverbeke DL, Hartson SD, Ramanathan R.Our objectives were to determine the non-enzymatic metmyoglobin reduction properties of bovine, porcine, and equine myoglobins and to characterize the effects of pre-incubation of 4-hydroxy-2-nonenal (HNE) with myoglobins on non-enzymatic metmyoglobin reduction in vitro. Purified bovine, porcine, and equine metmyoglobins (0.05 mM) were reduced at pH 5.6 and 7.4 in the presence or absence of HNE. Rates of metmyoglobin reduction were monitored by spectrophotometry, and myoglobin adducts were characterized by high-resolution mass-spectrometry. Results showed that the species origins of individual...
Saman E, Breugelmans K, Heyndrickx L, Merregaert J.The genome of equine infectious anemia virus (EIAV) contains several small open reading frames (ORFs), the importance of which in the development of the virus is not clear. We investigated the possibility that the largest of these ORFs (ORF S3) is expressed during the course of the viral infection. The ORF S3 information was expressed in Escherichia coli, and the antigen was used to raise monospecific antiserum. A 20-kDa protein expressed in cells producing EIAV was identified as the gene product of ORF S3. Furthermore, sera from EIAV-infected animals specifically recognized this protein, indi...
Pirhonen A, Linnala-Kankkunen A, Mënpää PH.P1 protamines isolated from ejaculated human, stallion, bull, boar and ram spermatozoa and P2 protamines from human and stallion spermatozoa were subjected, after alkaline phosphatase treatment, to in vitro phosphorylation reactions using cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). All P1 protamines were phosphorylated by PKA, whereas P2 protamines were phosphorylated only by PKC. In addition, human, stallion and boar, but not bull and ram, P1 protamines were phosphorylated by PKC. After phosphoamino acid analysis, the protamines showing positive signals for phosphoserine (...
Turki I, Hammami A, Kharmachi H, Mousli M.Human and equine rabies immunoglobulins are currently available for passive immunization against rabies. However, these are hampered by the limited supply and some drawbacks. Advances in antibody engineering have led to overcome issues of clinical applications and to improve the protective efficacy. In the present study, we report the generation of a trivalent single-chain Fv (scFv50AD1-Fd), that recognizes the rabies virus glycoprotein, genetically fused to the trimerization domain of the bacteriophage T4 fibritin, termed 'foldon' (Fd). scFv50AD1-Fd was expressed as soluble recombinant protei...
Coleridge M, Gillen AM, Farag R, Hanson RR, Munsterman AS.To investigate the knot holding capacity (KHC) of pattern-ending square, surgeon's and Aberdeen knots each tied in 4 throw combinations using large gauge suture after exposure to media commonly found in equine abdominal surgery. Methods: In vitro mechanical study. Methods: Knotted suture strands (n = 10/group). Methods: Strands of 2 polydioxanone or 3 polyglactin 910 were exposed to 1 of 4 media for 15 minutes. Control suture strands remained dry. Media used included balanced electrolyte solution, 1% sodium carboxymethylcellulose, equine serum, or equine fat. Pattern-ending knots for the 3...
