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.
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...
Veit M, Kabatek A, Tielesch C, Hermann A.Equine arteritis virus (EAV), a member of the newly established family Arteriviridae, is a small, positive-stranded RNA virus. It carries two protein complexes in its envelope, gp5/M and the recently described gp2b/gp3/gp4 complex. We report here on several basic features of EAV replication in cell culture and on the protein composition of virus particles. We have also characterized gp2b, gp3, and gp4 expressed using a baculovirus system in insect cells. Finally, we provide evidence that EAV possess hemagglutinating and hemolytic activity. The hemolysis assay might be useful for determining wh...
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...
Hoshino M, Kawata Y, Goto Y.GroEL interacts with proteins in denatured states and promotes their efficient folding. To understand the conformational features required for the substrate, we studied the interactions of GroEL with various derivatives of horse cytochrome c including porphyrin-cytochrome c, apo-cytochrome c, and the three fragments containing the heme group, i.e. fragments 1-65, 1-38 and 11-21. Size-exclusion chromatography was performed, taking advantage of the heme absorption of the fluorescence label. Under low-salt conditions, significant binding to GroEL was observed for porphyrin-cytochrome c, apo-cytoc...
Haerdi-Landerer MC, Suter MM, Steiner A, Wittenbrink MM, Pickl A, Gander BA.For the treatment of septic arthritis in large animals, the local application of antibiotics as a slow release system may be an appropriate means to reach high local bioactivity and low systemic side effects and drug residues. In this study, doxycycline microspheres were developed and tested in vitro for their drug-release properties, suitability for intra-articular application and antimicrobial activity. Methods: The development of a slow release system was achieved by microencapsulation of the drug into poly(lactide-co-glycolide) microspheres by a novel ultrasonic atomization method. Drug el...
Dufour P, Courtois J, Seynaeve Y, Peeters S, Le Goff C, Cavalier E, Ponthier J.Steroid concentrations in serum are fluctuating during pregnancy of many mammal species. The current knowledge about endocrinology of gestation is mainly based on immunoassays. However, the lack of specificity of these assays hampers the reliability of the results. In the present work, we developed and validated a methodology associating liquid chromatography (LC) and mass spectrometry (MS) to simultaneously quantify, with high specificity and accuracy, estrone-3-sulfate (E3S), progesterone (PRO), estrone (E1) and estradiol (E2) in serum of two different mammal species. The sample preparation ...
Nugraheni AD, Nagao S, Yanagisawa S, Ogura T, Hirota S.We have previously shown that methionine-heme iron coordination is perturbed in domain-swapped dimeric horse cytochrome c. To gain insight into the effect of methionine dissociation in dimeric cytochrome c, we investigated its interaction with cyanide ion. We found that the Soret and Q bands of oxidized dimeric cytochrome c at 406.5 and 529 nm redshift to 413 and 536 nm, respectively, on addition of 1 mM cyanide ion. The binding constant of dimeric cytochrome c and cyanide ion was obtained as 2.5 × 10(4) M(-1). The Fe-CN and C-N stretching (ν (Fe-CN) and ν (CN)) resonance Raman ba...
Heydrick FP, Comer JF, Wachter RF.Phospholipid analyses of Venezuelan equine encephalitis virus showed that virus propagated in L-cell monolayers had a higher sphingomyelin content and a lower phosphatidylcholine content than virus grown in chick fibroblast monolayers. Virus of L-cell origin also was found to possess greater thermal stability than virus derived from the chick fibroblast cell.
Stojan J.Highly efficient and rapid lead compound evaluation for estimation of inhibition parameters and type of inhibition is proposed. This is based on a single progress-curve measurement in the presence of each candidate compound, followed by the simultaneous analysis of all of these curves using the ENZO enzyme kinetics suite, which can be implemented as a web application. In the first step, all of the candidate ligands are tested as competitive inhibitors. Where the theoretical curves do not correspond to the experimental data, minimal additional measurements are added, with subsequent processing ...
Gamoh K, Nakamura S.Japan established a vaccine selection system, in which a committee evaluates veterinary influenza vaccines to determine if the vaccine should be updated. In 2013, it was concluded that the present equine influenza vaccine strains did not have to be updated, but clade 2 (Fc2) viruses of the Florida sublineage should be included. We collected three Fc2 viruses as candidates and conducted comparative tests. Results indicated that A/equine/Carlow/2011 (H3N8) is not suitable, because of its unstable antigenic characteristics. A comparison between A/equine/Richmond/1/2007 (H3N8) (Richmond/07) and A/...
Kordula T, Dubin A, Schooltink H, Koj A, Heinrich PC, Rose-John S.Horse blood leucocytes contain an elastase inhibitor (HLEI) belonging to the serpin family. Poly(A)+RNA isolated from these cells was used to construct a cDNA library in lambda gt10, which was first screened with a synthetic degenerate oligonucleotide probe corresponding to the amino acid sequence of the reactive centre of the inhibitor. Three clones were obtained covering the entire coding region of the protein. Sequencing of these clones showed identity with the amino acid sequence obtained from Edman degradation of the elastase inhibitor. The coding sequence of the HLEI cDNA was cloned into...
Haque RU.A technique for identifying and characterizing staphylococcal hemolysins by first separating them electrophoretically in barbital-buffered agar gel (pH 8.4) at 5 ma/cm for 2 hr and then determining their hemolytic activities by exposing them to human, horse, rabbit, and sheep erythrocytes is described. The alpha-hemolysin produced by a White variant of the Wood 46 strain of Staphylococcus aureus migrated 18 mm towards the cathode, and it lysed horse, rabbit, and sheep erythrocytes, whereas a Clear variant of the Wood 46 strain of S. aureus produced a lysin which migrated similarly to the alpha...
Kavak A, Johannisson A, Lundeheim N, Rodriguez-Martinez H, Aidnik M, Einarsson S.Methods to evaluate the quality of frozen-thawed stallion semen are still needed, particularly those considering the sperm function. The present study evaluated sperm motility, membrane and acrosome integrity and the capacitation status of frozen-thawed spermatozoa from seven Tori and six Estonian breed stallions by way of computer assisted sperm analysis (CASA), a triple fluorophore stain combination and Merocyanine 540, respectively, the latter ones using flow cytometry. Two ejaculates from each stallion were cryopreserved using the Hannover method in 0.5 ml plastic straws. Two straws per ej...
Lunn DP, Holmes MA, Schram B, Duffus WP.Equine immunoglobulin G is currently classified as consisting of five sub-isotypes: IgGa, b, and c, IgG(T), and IgG(B). The study of the role of these immunoglobulins in antigen-specific responses, and the examination of their functional properties would be greatly facilitated by the availability of monoclonal antibodies (Mabs) that distinguish between them. The production and characterization of two Mabs that recognize an IgG sub-isotype with the characteristics of IgG(ab) is described. The immunoglobulin identified by these Mabs had a heavy chain weight of 53 kDa, was of rapid cathodal elect...
Weitnauer E, Robitzki A, Layer PG.Several side activities have been attributed to butyrylcholinesterase (BChE), including aryl acylamidase (AAA) activity, which is an amidase-like activity with unknown physiological function splitting the artificial substrate o-nitroacetanilide. For avians, extensive developmental data have pointed to neurogenetic functions of BChE, however, a possible AAA activity of BChE has not been studied. In this study, we first compare the relative levels of AAA exhibited by BChE in whole sera from chick, fetal calves (FCS) and horse. Remarkably, FCS exhibits a 400-fold higher ratio of AAA/BChE than hor...
Nie GJ, Wenzel JG.Hypoosmotic swelling (HOS) is used for assessing plasma membrane function and fertilizing capacity of human spermatozoa. However, HOS solutions and methodologies have not been evaluated specifically for assessing stallion spermatozoa. The objective of this study was to identify a HOS solution and assay conditions specifically for stallions that would maximize spermatozoal plasma membrane swelling. The HOS solutions and assay conditions, including incubation time (15 to 180 min), temperature (25 degrees vs 37 degrees C), and total number of cells examined (100, 200 or 500) were evaluated. Assay...
Casaite V, Bruzyte S, Bukauskas V, Setkus A, Morozova-Roche LA, Meskys R.Equine lysozyme (EL) is a calcium (Ca)-binding lysozyme and is an intermediary link between non-Ca-binding C-type lysozyme and alpha-lactalbumin. The feature of lysozymes to assemble into the fibrils has recently gained considerable attention for the investigation of the functional properties of these proteins. To study the structural and functional properties of EL, a synthetic gene was cloned and EL was overexpressed in Escherichia coli as a fused protein. The His-tagged recombinant EL was accumulated as inclusion bodies. Up to 50 mg/l of the recombinant EL could be achieved after purificati...
Beckelmann J, Budik S, Bartel C, Aurich C.Until now, sex determination in equine embryos has been performed by detection of Y-chromosome-specific sequences only. In the present study, expression of a Barr-body-specific marker, the X-inactivated-specific transcript (Xist) gene, whose gene product consists of RNA which coats and thereby inactivates one of the X chromosomes, was investigated in equine embryos produced in vivo. Preattachment embryos at different times after ovulation (Day 8: n = 9; Day 10: n = 12; Day 12: n = 15) were analyzed for Xist RNA expression using quantitative and qualitative reverse transcription-polymerase chai...
Mirajkar NS, Kelley MR, Gebhart CJ.Reported herein is the draft genome sequence of equine-origin Lawsonia intracellularis strain E40504, an obligate intracellular bacterium and the etiological agent of equine proliferative enteropathy. The 1.69-Mb draft genome sequence includes 1,380 protein-coding genes and 49 RNA genes, and it lacks a genomic island reported in swine-origin L. intracellularis strain PHE/MN1-00.
Singh RK, Kooreman KM, Babbs CF, Fessler JF, Salaris SC, Pham J.The scavenging of superoxide radicals by endogenous and therapeutically administered superoxide dismutases may prevent superoxide-mediated oxidative stress leading to lipid peroxidation, membrane lysis, and cell death in a wide variety of normal and pathologic states. Simple inorganic manganous salts such as MnCl2 also have superoxide dismutase-like activity and are extremely inexpensive, compared with enzymatic superoxide dismutase preparations. In this study, we explored the use of Mn salts as antioxidant drugs. We used the percentage of inhibition of nitroblue tetrazolium reduction by super...
van Woensel PA, Goovaerts D, Markx D, Visser N.A mouse model was developed for testing the pathogenicity of equine herpes virus-1 (EHV-1) strains. The model was validated with EHV-1 strains that are known to be of a low or high pathogenicity in horses. From all parameters tested, the safety index, which was calculated from the body weights of the mice after infection, proved to be the best predictive parameter. When this parameter was used, good and reliable correlations were found with the pathogenicity of the EHV-1 strains in horses. This method enabled the differentiation between the two experimental EHV-1 strains whose genetic backgrou...
Wattrang E, Berg M, Magnusson M.This study aimed to evaluate different nucleic acid preparations as cytokine inducers in equine cells. To induce cytokine production, bacterial plasmid DNA or short synthetic oligodeoxyribonucleotides (ODN), with or without the transfection reagent lipofectin, were added to cultures of purified equine peripheral blood mononuclear cells (PBMC). Cytokine activity was detected with bioassays in cell culture supernatants after 24h of induction and cytokine mRNA expression was detected using RT-PCR at 6h post induction. For IFN-alpha/beta it was found that both plasmid DNA and phosphodiester ODN, c...
Clabough DL, Conboy HS, Roberts MC.Using radial immunodiffusion as a standard, 4 screening techniques for detection of failure of passive transfer in equine neonates were compared for sensitivity, specificity, positive and negative predictive values, efficiency, and cost. The techniques compared were latex agglutination test, membrane filter ELISA, dipstick ELISA, and glutaraldehyde coagulation (GC) test. Test results of 50 serum samples from foals 24 to 60 hours old revealed consistently highest accuracy in the GC test at IgG concentrations of 400 and 800 mg/dl, and lowest cost per test, using the GC test. Two hundred fifty-th...
Foldager C, Bendtsen M, Nygaard JV, Zou X, Bünger C.To investigate the microstructural differences and responsible mechanisms in early bone formation in anterior lumbar interbody fusion (ALIF) in the spine using rhBMP-2 (INFUSE), equine bone protein extract (COLLOSS E) or autograft. Methods: Twelve Danish female landrace pigs underwent a 3-level ALIF procedure at L3-6. PEEK interbody cages packed with rhBMP-2, COLLOSS E, or autograft were inserted. The animals were divided into two groups of six, and observed for four and eight weeks postoperatively. MicroCT was performed for evaluation of microstructure of the bone within the cage. A mathemati...
Strange R, Morante S, Stefanini S, Chiancone E, Desideri A.Extended X-ray absorbance fine structure measurements have been carried out on the initial Fe(III)-apoferritin complex at a Fe/subunit ratio of 2 in native and modified horse spleen apoferritin. Analysis of the data indicates that in the native protein the iron forms a protein-bound polynuclear cluster (Fe-Fe distance 3.4 A) with a first coordination sphere constituted by 5-6 low-Z atoms, e.g., nitrogen atoms, carboxylate-like ligands or oxo bridges between the iron atoms. Modification of Cys-126, a residue localized on the outer surface of the hydrophilic three-fold channels, with p-chloromer...
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...
Siles-Lucas M, Cuesta-Bandera C.A comparison was made, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), of excretory/secretory (ES)-crude and immunopurified (with the corresponding anti-host serum) hydatid fluids-and somatic (S)-protoscoleces-proteins, from several ovine, equine, swine, bovine and human Echinococcus granulosus Spanish isolates. Likewise, the host influence on parasitic ES protein expression was studied, comparing purified hydatid fluids from ovine and equine cysts obtained from natural hosts and in RNMI mice. Purified hydatid fluids patterns, under reducing conditions, yielded the mos...
Sander J, Terhardt M, Sander S, Aboling S, Janzen N.Up to now quantification of hypoglycin A in serum and urine in the range of nmols to μmols per liter plus the measurement of accumulated acyl conjugates have been used for the diagnosis of poisoning by fruits or seeds of in humans and animals. A second poison, methylenecyclopropylglycine, however, is known to occur in this material. The objective of our study was to develop and evaluate a method for the quantification of this compound suitable for test materials obtained from animals and man. Methods: Methylenecyclopropylglycine was extracted from serum and urine of a volunteer by a methanoli...
Jo A, Lee HE, Kim HS.Horses (Equus caballus) have been domesticated and bred to enhance speed, strength, and agility. Members of the Equus caballus Endogenous Retrovirus (EqERV) family affect several of these abilities in horses. EqERV elements have been integrated in the horse genome during evolution and generate repeat elements such as long terminal repeats (LTRs). LTR sequences are involved in retrovirus replication and play an essential function in post-transcriptional control mechanisms, such as by providing binding sites for microRNAs (miRNAs) or generating miRNA precursors. In this study, we identified a no...
Lin J, Merryweather J, Vitello LB, Erman JE.The kinetics of formation and dissociation of the horse metmyoglobin/azide complex has been investigated between pH 3.5 and 11.5. The ionic strength dependence of the reaction has been determined at integral pH values between 5 and 10. Hydrazoic acid, HN3, binds to metmyoglobin with a rate constant of (3.8 +/- 1.0) x 10(5) M-1 s-1. Protonation of a group with an apparent pKa of 4.0 +/- 0.3 increases the rate of HN3 binding 6.5-fold to (2.5 +/- 0.8) x 10(6) M-1 s-1. The ionizable group is attributed to the distal histidine, His-64. The azide anion, N-3, binds to metmyoglobin with a rate constan...
Fitzgibbon JE, Sagripanti JL.The development of a method in macroarray format for the identification of alphaviruses and orthopoxviruses in samples of concern in biodefense is reported. Capture oligonucleotides designed to bind generic members of the orthopox- or alphavirus families and a collection of additional oligonucleotides to bind specifically nucleic acids from five individual alphaviruses, including Venezuelan equine encephalitis, or DNA from each of four orthopoxviruses, including variola virus (VAR) were deposited onto nylon membranes. Hybridization of digoxigenin labeled PCR products to the macroarray produced...
