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.
Suagee JK, Corl BA.Culturing adipocytes enables fine control of experimental conditions and helps minimise animal use. This report describes an explant-based method for isolating stromal-vascular cells from equine adipose tissue that enables use of small amounts of tissue. Subcutaneous and mesenteric adipose tissues were harvested post mortem and stromal-vascular cells grown from explants, prior to testing the capacity of several differentiation media to induce lipid droplet formation and increase transcript abundance of adipocyte markers. Inclusion of rosiglitazone at 1 and 5 µmol/l concentrations, along with ...
Pisano MB, Seco MP, Ré VE, Farías AA, Contigiani MS, Tenorio A.Venezuelan Equine Encephalitis (VEE) complex belongs to alphavirus genus in the family Togaviridae. Several species of this complex are pathogenic to humans. VEE infections can produce severe or mild disease, and many cases remain undiagnosed. A specific and sensitive reverse transcriptase nested polymerase chain reaction (RT-Nested PCR) method was developed for the detection of all VEE subtypes, including Rio Negro Virus (RNV) (subtype VI), which circulates only in Argentina. Degenerated primers were designed and thermal cycling parameters were standardized. This technique is suitable for rap...
Wang CC, Hartmann-Fischbach P, Krueger TR, Wells TL, Feineman AR, Compton JC.3,4-Methylenedioxypyrovalerone (MDPV) is a psychoactive drug with potent stimulant properties and potential for abuse and drug dependency. MDPV was recently classified as a Class I drug by Racing Commissioners International, indicating that it is a banned substance in equine athletes because it lacks therapeutic value in horses. To enforce this ban, a sensitive and fast liquid chromatography-tandem mass spectrometry method was needed. It is for this reason that this method was developed for quantification and confirmation of MDPV in equine plasma. Sample preparation involved liquid-liquid extr...
Vidament M, Magistrini M, Le Foll Y, Levillain N, Yvon JM, Duchamp G, Blesbois E.This study tested whether variable temperatures (from -0.5 to 15 °C) and air exposure could be used under laboratory and under field conditions to store stallion sperm diluted in extender INRA96 without loss of fertility. Experiment 1 (laboratory conditions) measured the effects of two 72 h storage conditions (5 °C with air vs. 15 °C without air). Experiment 2 (fixed field conditions) measured the effects of 22 h of storage without air in disposable containers maintained at four ambient temperatures (7 °C, 17 °C, 27 °C, 39 °C with semen at -0.5 °C to 3 °C, 4 °C to 7 °C, 8 °C to 10 ...
Kim DS, Kim DW, Kim MY, Nam SH, Choi SH, Kim RN, Kang A, Kim A, Park HS.A conjoined gene is defined as one formed at the time of transcription by combining at least part of one exon from each of two or more distinct genes that lie on the same chromosome, in the same or opposite orientation, which translate independently into different proteins. We comparatively studied the extent of conjoined genes in thirteen genomes by analyzing the public databases of expressed sequence tags and mRNA sequences using a set of computational tools designed to identify conjoined genes on the same DNA strand or opposite DNA strands of the same genomic locus. The CACG database, avail...
Sahmi F, Nicola E, Price CA.The objective was to establish a cell line-based bioassay for FSH in horse serum for screening samples with high eCG bioactivity. A cell line (HEK293) was transiently cotransfected with an FSH reporter expression plasmid and a cAMP-responsive β-galactosidase reporter plasmid. Cells were bulk frozen, and thawed for assay purposes. This assay was specific for FSH, with no cross-reaction with LH or insulin-like growth factor-1. Standard curves (eCG) and serum samples from pregnant mares passed parallel line bioassay validity tests (linearity and parallelism). Estimates of bioactivity with this b...
Janda J, Plattet P, Torsteinsdottir S, Jonsdottir S, Zurbriggen A, Marti E.Allergic horses react to innocuous environmental substances by activation of Th2 cells and production of allergen-specific IgE antibodies. The mechanisms leading to Th2 differentiation are not well understood. In humans and mice, epithelial cell-derived thymic stromal lymphopoietin (TSLP) plays a central role in this process. Little is known about equine TSLP (eqTSLP) and its role in allergic diseases and our current knowledge is limited to the assessment of TSLP mRNA expression. In order to be able to study eqTSLP at the protein level, the aim of the present study was to produce recombinant e...
Soares JH, Brosnan RJ, Fukushima FB, Hodges J, Liu H.Anesthetic blood solubility predicts pharmacokinetics for inhaled agents and is essential for determination of blood anesthetic concentrations from end-tidal gas concentrations using Henry's Law. Though used to model anesthetic effects in humans, there are limited interspecies solubility comparisons that include modern haloethers. This study aimed to measure hematocrit-adjusted blood:gas anesthetic partition coefficients (λ B:G) for desflurane, sevoflurane, isoflurane, and methoxyflurane in humans and animals. Methods: Whole blood was collected from 20 rats, 8 horses, and 4 each of cats, catt...
Boyle AG, Boston RC, O'Shea K, Young S, Rankin SC.Streptococcus equi is the etiologic agent of a highly infectious upper respiratory disease of horses known as strangles. Bacterial culture methods and polymerase chain reaction (PCR) of nasopharyngeal washes and guttural pouch lavages are used routinely to test clinical and carrier animals for the presence of S. equi but no definitive or gold standard test method has been shown to be optimal. We hypothesized that (i) a flocked swab submerged in ten-fold serial dilution suspensions of S. equi prepared in 0.9% NaCl would detect more colony forming units (CFU) than a rayon swab when used to inocu...
Gray BP, Biddle S, Pearce CM, Hillyer L.Fluticasone propionate (FP) is an anti-inflammatory agent with topical and inhaled applications commonly used in the treatment of asthma in steroid-dependent individuals. The drug is used in racehorses to treat Inflammatory Airway Disease; this work was performed in order to advise on its use and detect potential misuse close to racing. Methods were developed for the extraction and analysis of FP from horse plasma and a carboxylic acid metabolite (FP-17βCOOH) from horse urine. The methods utilize ultra high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) in ...
Lönnberg M, Bondesson U, Cormant F, Garcia P, Bonnaire Y, Carlsson J, Popot MA, Rollborn N, Råsbo K, Bailly-Chouriberry L.Doping of horses with recombinant human erythropoietin (rHuEPO) to illegally enhance their endurance capacity in horseracing has been reported during the last years. This leads to increased blood viscosity which can result in sudden death and is of concern for the horse welfare. Additionally, the horse can start production of rHuEPO antibodies, which cross-reacts with endogenous equine EPO and can lead to severe anaemia and even death. In this study, a novel micro-chromatographic method, EPO WGA MAIIA, has been tested for the capability in plasma and urine samples to detect administration of e...
Cwiklinski K, Kooyman FN, Van Doorn DC, Matthews JB, Hodgkinson JE.Cyathostomins comprise a group of 50 species of parasitic nematodes that infect equids. Ribosomal DNA sequences, in particular the intergenic spacer (IGS) region, have been utilized via several methodologies to identify pre-parasitic stages of the commonest species that affect horses. These methods rely on the availability of accurate sequence information for each species, as well as detailed knowledge of the levels of intra- and inter-specific variation. Here, the IGS DNA region was amplified and sequenced from 10 cyathostomin species for which sequence was not previously available. Also, add...
Liu C, Guo W, Lu G, Xiang W, Wang X.A new strain of equine herpesvirus type 8 (EHV-8), Wh, has been isolated from horses in China, and its complete genome has been sequenced and analyzed. The result indicates that the new strain has the same constitution and arrangement of open read frames as EHV-1 and EHV-9. This work is the first announced complete genome sequence of EHV-8.
Macias Rioseco M, Beingesser J, Uzal FA.Clostridium perfringens type C causes necrotizing enteritis mostly in neonatal animals of several species, including horses. The virulence of C. perfringens type C is mostly mediated by beta toxin (CPB). This toxin is highly sensitive to the action of trypsin and other proteases, which explains the increased susceptibility of neonatal animals to type C infections. Final confirmation of type C disease diagnosis should be based on detection of CPB in the intestinal content of affected animals. However, because CPB is so sensitive to the action of proteases, it is believed that this toxin persist...
Riond B, Hofmann-Lehmann R, Lutz H.A point-of-care device (POCD) for measuring total white blood cell count was evaluated for feline, canine, equine and bovine blood samples collected into EDTA. Mean biases were -9.2% (range, -12% to -6.3%) for feline samples, 20.2% (range, 15.3-25.1%) for canine samples, -7.1% (range, -8.3% to -5.9%) for equine samples, and 0.7% (range, -1.1% to 2.5%) for bovine samples. The results were influenced by the presence of nucleated red blood cells. The POCD provided precise, reliable data for feline, equine and bovine samples but the values obtained for the canine counts were overestimations.
Ronchi GF, Ulisse S, Rossi E, Franchi P, Armillotta G, Capista S, Peccio A, Di Ventura M, Pini A.Monovalent, inactivated and adjuvanted vaccines against African horse sickness, prepared with serotypes 5 and 9, were tested on guinea-pigs to select the formulation that offered the greatest immunity. The final formulation of the vaccines took into account the immune response in the guinea-pig and the inflammatory properties of two types of adjuvant previously tested on target animals. A pilot study was subsequently conducted on horses using a vaccine prepared with serotype 9. The vaccine stimulated neutralising antibodies from the first administration and, after the booster dose, 28 days lat...
Xijier , Mori Y, Fukuoka M, Cairangzhuoma , Inagaki M, Iwamoto S, Yabe T, Kanamaru Y.Native alpha-lactalbumins (α-LA) from equine, bovine, and human milk were not cytotoxic. However, after treatment with trifluoroethanol (TFE), all three α-LAs exhibited cytotoxicity. Toxic potencies were distinctly different among them. Equine α-LA was the most robust, bovine α-LA was moderate, and human α-LA was weak. There were no significant structural changes as between the native and the TFE-treated α-LAs.
Ababneh MM, Troedsson MH.The aim of this study was to determine phospholipase A2 (PLA2) kinetics and activity in the mare's endometrium during the oestrous cycle and early pregnancy. Phospholipase A2 is responsible for the liberation of arachidonic acid from phospholipids, which is the first limiting step in prostaglandins synthesis. Phospholipase A2 activity was measured using an assay based on the liberation of oleic acid from 1-palmitoyl-2-[(14) C] oleoyl phosphatidylcholine. The enzyme was shown to be calcium dependent, to have an optimum pH of 8 and an apparent Michaelis constant of 127 μM. Enzyme activity was l...
Macías-García B, González-Fernández L, Gallardo-Bolaños JM, Peña FJ, Johannisson A, Morrell JM.The aim of this study was to elucidate if SLC after 24 h storage selects the subpopulation of spermatozoa that better withstands osmotic shock. To test this hypothesis, viability, mitochondrial membrane potential (MMP) and superoxide anion (O(2)(·-)) production of uncentrifuged (UC) and single layer centrifugation (SLC) - selected spermatozoa were analyzed following SLC after storage of the semen. An aliquot of the extended ejaculate (100×10(6) spermatozoa/mL) was centrifuged through a single layer of a silane-coated silica based colloid formulation optimized for equine spermatozoa (Androcol...
Burbelo PD, Dubovi EJ, Simmonds P, Medina JL, Henriquez JA, Mishra N, Wagner J, Tokarz R, Cullen JM, Iadarola MJ, Rice CM, Lipkin WI, Kapoor A.Genetic and biological characterization of new hepaciviruses infecting animals contributes to our understanding of the ultimate origins of hepatitis C virus (HCV) infection in humans and dramatically enhances our ability to study its pathogenesis using tractable animal models. Animal homologs of HCV include a recently discovered canine hepacivirus (CHV) and GB virus B (GBV-B), both viruses with largely undetermined natural host ranges. Here we used a versatile serology-based approach to determine the natural host of the only known nonprimate hepacivirus (NPHV), CHV, which is also the closest p...
Tallmadge RL, Such KA, Miller KC, Matychak MB, Felippe MJ.Common variable immunodeficiency (CVID) is a heterogeneous disorder of B cell differentiation or function with inadequate antibody production. Our laboratory studies a natural form of CVID in horses characterized by late-onset B cell lymphopenia due to impaired B cell production in the bone marrow. This study was undertaken to assess the status of B cell differentiation in the bone marrow of CVID-affected horses by measuring the expression of genes essential for early B cell commitment and development. Standard RT-PCR revealed that most of the transcription factors and key signaling molecules ...
Bailly-Chouriberry L, Cormant F, Garcia P, Lönnberg M, Szwandt S, Bondesson U, Popot MA, Bonnaire Y.Recombinant human erythropoietin (rHuEPO) is a 30-34 kDa glycoprotein banned by the racing authorities. For some years this molecule has been detected in race horses in USA and in Europe, and even in racing camels. Although direct methods to differentiate horse endogenous EPO and rHuEPO have been developed either by LC-MS/MS or by isoelectric focusing (IEF) with double-blotting, the short confirmation time of such prohibited hormone in plasma remains a problem for horseracing doping control laboratories. In order to improve the rHuEPOs confirmation process in horse plasma or urine in terms of ...
Fuchs S, Klier J, May A, Winter G, Coester C, Gehlen H.Delivering active ingredients using biocompatible and biodegradable carriers such as gelatin nanoparticles (GNPs) to the lung constitutes a promising non-invasive route of administration. However, the pulmonary delivery of nanoparticle-based immunotherapy is still a field that requires more clarification. In this study, GNPs loaded with cytosine-phosphate-guanine oligodeoxynucleotides (CpG-ODN)-loaded and plain GNPs were aerosolised either by a conventional pressured metered dose inhaler (pMDI) or by active or passive vibrating-mesh (VM) nebulisers. GNP sizes after nebulisation by active and p...
Kraus MS, Kaufer BB, Damiani A, Osterrieder N, Rishniw M, Schwark W, Gelzer AR, Divers TJ.To date, no information is available on the true biological elimination half-life (T(1/2) ) of cardiac troponin I (cTnI) in the equine species. Such data are required to better evaluate the optimal time to acquire the cTnI sample following acute myocardial injury. Objective: To determine the T(1/2) of equine cTnI. Methods: Four healthy ponies received i.v. injections of recombinant equine cTnI. Plasma cTnI concentrations were measured with a point-of-care cTnI analyser at multiple time points after injection. Standard pharmacokinetic analysis was performed to establish the T(1/2) of cTnI. Resu...
Coutinho da Silva MA, Seidel GE, Squires EL, Graham JK, Carnevale EM.The effects of semen extender components on the ability of stallion sperm to bind to the zona pellucida (ZP) and the suitability of using bovine ZP for a ZP-binding assay for stallion sperm were investigated in a series of experiments. In Experiment I, binding of stallion sperm to both bovine and equine ZP was significantly increased when a skim milk-based extender (EZM) was used. In Experiment II, a threefold increase in sperm binding to ZP was observed when sperm were diluted in EZM compared with diluents, which contained no milk (TALP, LAC, and EmCare). In Experiment III, centrifuging the s...
Kavar T, Čeh E, Dovč P.Coat color of gray horses is associated with a 4.6-kb duplication, which can be determined using PCR amplification of about 5-kb DNA fragment. In practice, this means that amplification might fail frequently. Therefore, a novel genetic screening method based on amplification of the 246 bp DNA fragment has been developed.
Wulijideligen , Asahina T, Hara K, Arakawa K, Nakano H, Miyamoto T.The purification and characterization of a bacteriocin produced by Leuconostoc mesenteroides strain 406 that was isolated from traditional Mongolian fermented mare's milk, airag, were carried out. Leuconostoc mesenteroides strain 406 was identified on the basis of its morphological and biochemical characteristics and carbohydrate fermentation profile and by API 50 CH kit and 16S ribosomal DNA analyses. The neutral-pH cell-free supernatant of this bacterium inhibited the growth of several lactic acid bacteria and food spoilage and pathogenic organisms, including Listeria monocytogenes and Clost...
Fidani M, Pompa G, Mungiguerra F, Casati A, Fracchiolla ML, Arioli F.After the detection of low concentrations of prednisolone in racehorse urine samples collected at Italian racetracks, a study was initiated to investigate the accuracy of the analytical protocol used and the possible endogenous origin of detected prednisolone. Methods: Multiple reaction monitoring (MRM) MS(2) acquisition with a triple quadrupole (n = 780) and full scan MS(2) and MS(3) (n = 180) acquisition with a linear ion trap were checked. As a further confirmation, ten urine samples were analysed by high-resolution mass spectrometry (HRMS). Results: The study showed the difficulty of ident...
Pennick KE, McKnight CA, Patterson JS, Latimer KS, Maes RK, Wise AG, Kiupel M.Immunohistochemistry (IHC) and in situ hybridization (ISH) can be used either to detect or to differentiate between Eastern equine encephalitis virus (EEEV) and West Nile virus (WNV) within formalin-fixed, paraffin-embedded (FFPE) brain tissue of horses. To compare the diagnostic sensitivity and specificity of ISH and IHC, FFPE brain tissue from 20 EEEV-positive horses and 16 WNV-positive horses were tested with both EEEV and WNV oligoprobes and EEEV- and WNV-specific antibodies. Reverse transcription polymerase chain reaction (RT-PCR) for detection of EEEV and WNV was used as the gold standar...
Albrizio M, Lacalandra GM, Micera E, Guaricci AC, Nicassio M, Zarrilli A.Opioid receptors and endogenous opioid peptides act not only in the control of nociceptive pathways, indeed several reports demonstrate the effects of opiates on sperm cell motility and morphology suggesting the importance of these receptors in the modulation of reproduction in mammals. In this study we investigated the expression of delta opioid receptors on equine spermatozoa by western blot/indirect immunofluorescence and its relationship with sperm cell physiology. Methods: We analyzed viability, motility, capacitation, acrosome reaction and mitochondrial activity in the presence of naltri...
Handschuh S, Okada CTC, Walter I, Aurich C, Glösmann M.Here, we describe a workflow for high-detail microCT imaging of formalin-fixed and paraffin-embedded (FFPE) equine embryos recovered on Day 34 of pregnancy (E34), a period just before placenta formation. The presented imaging methods are suitable for large animals' embryos with intention to study morphological and developmental aspects, but more generally can be adopted for all kinds of FFPE tissue specimens. Microscopic 3D imaging techniques such as microCT are important tools for detecting and studying normal embryogenesis and developmental disorders. To date, microCT imaging of vertebrate e...
Todd EW.Antiproteinase sera were prepared by immunizing horses with filtrates from a selected strain of group A streptococcus. This strain, which produced high titred proteinase but no erythrogenic toxin, was selected from forty-two strains of group A streptococci which produced varying amounts of proteinase. A few strains belonging to groups B, C, and G were also tested; they were all proteinase-negative. Methods are described for titrating streptococcal proteinase in crude culture filtrates and for measuring the antiproteinase activity of serum. The antiproteinase titres of sera from immunized horse...
Rossi TM, Kavsak PA, Maxie MG, Pearl DL, Pyle WG, Physick-Sheard PW.Human cardiac troponin I (cTnI) assays have been used in equine medicine, often without prior analytical validation for equine use. In the absence of appropriate validation, the clinical significance of assay results is uncertain and can lead to misdiagnosis. We followed the American Society for Veterinary Clinical Pathology guidelines and investigated linearity, precision, limit of quantification (LoQ), and comparative recovery for 6 commercial cTnI assays developed for use in human medicine. Clinically acceptable linearity was observed in assays A-D, whereas assay E did not detect equine cTn...
Metz GE, Abeyá MM, Serena MS, Panei CJ, Echeverría MG.Equine arteritis virus (EAV) induces apoptosis in infected cells. Cell death caused by EAV has been studied mainly using three cell lines, BHK-21, RK-13 and Vero cells. The mechanism of apoptosis varies among cell lines and results cannot be correlated owing to differences in EAV strains used. We evaluated different markers for apoptosis in BHK-21, RK-13 and Vero cell lines using the Bucyrus EAV reference strain. Acridine orange/ethidium bromide staining revealed morphological changes in infected cells, while flow cytometry indicated the extent of apoptosis. We also observed DNA fragmentation,...
Valle-Casuso JC, Gaudaire D, Martin-Faivre L, Madeline A, Dallemagne P, Pronost S, Munier-Lehmann H, Zientara S, Vidalain PO, Hans A.RNA viruses are responsible for a large variety of animal infections. Equine Arteritis Virus (EAV) is a positive single-stranded RNA virus member of the family Arteriviridae from the order Nidovirales like the Coronaviridae. EAV causes respiratory and reproductive diseases in equids. Although two vaccines are available, the vaccination coverage of the equine population is largely insufficient to prevent new EAV outbreaks around the world. In this study, we present a high-throughput in vitro assay suitable for testing candidate antiviral molecules on equine dermal cells infected by EAV. Using t...
Weiss DJ, Evanson OA.To evaluate lipopolysaccharide (LPS)-induced activation of equine neutrophils in blood. Methods: Blood samples from 5 healthy adult Thoroughbreds. Methods: Neutrophil integrin (CD11/CD18) expression, size variation, degranulation, and deformability were measured with and without incubation with LPS. Time and concentration studies were done. The mechanism of endotoxin-induced neutrophil activation was investigated by inactivating complement or preincubating neutrophils with inhibitors of tumor necrosis factor-alpha (TNF-alpha) synthesis, prostaglandin-leukotriene synthesis, or platelet-activati...
Vukojević V, Bowen AM, Wilhelm K, Ming Y, Ce Z, Schleucher J, Hore PJ, Terenius L, Morozova-Roche LA.Recent evidence supports the idea that early aggregates, protein, and lipoprotein oligomers but not large aggregates like fibrils that are formed at late stages of the aggregation process are responsible for cytotoxicity. Oligomers can interact with the cellular plasma membrane affecting its structure and/or dynamics or may be taken up by the cells. In either case, disparate cascades of molecular interactions are activated in the attempt to counteract the disturbance induced by the oligomers. If unsuccessful, cell death follows. Here, we study the molecular and cellular mechanisms underlying P...
Oke SL, Hurtig MB, Keates RA, Wright JR, Lumsden JH.To determine whether 3 variations of the 1,9-dimethylmethylene blue (DMMB) assay yield comparable results when measuring sulfated glycosaminoglycan (sGAG) concentrations in equine synovial fluid (SF). Methods: 25 samples of SF collected from affected joints of 13 horses and 13 samples of SF collected from nonaffected (control) joints of 4 horses. Methods: Sulfated glycosaminoglycan concentrations were measured by the direct spectrophotometric (ie, Farndale), microplate, and indirect DMMB assays in samples of SF collected from normal and affected joints and in samples digested with nucleases, p...
Arata AB, Cooke CL, Jang SS, Hirsh DC.It is difficult to distinguish isolates of Taylorella equigenitalis, the cause of contagious equine metritis, from a T. equigenitalis-like organism isolated from asymptomatic donkeys and horses. Although T. equigenitalis is responsible for a severe, contagious disease of the reproductive tract of equids, the T. equigenitalis-like organism, although contagious, does not appear to produce disease. Because of the economic consequences of correctly distinguishing isolates of these 2 microorganisms, a polymerase chain reaction (PCR)-based assay was developed that will distinguish isolates of T. equ...
Raeside JI, Renaud RL, Christie HL.Dehydroepiandrosterone (DHEA) and 3 beta-hydroxyandrosta-5,7-dien-17-one (7-dehydro-DHEA) are secreted in large quantities by the remarkably hypertrophied fetal gonads of both sexes in the pregnant mare. Their secretion serves as the fetal component of a feto-placental unit for oestrogen production in equine pregnancies. They are secreted in large amounts but show a decline in late pregnancy when the fetal gonads regress and levels of oestrogens in the mare fall as a consequence. We have examined the levels of these precursor steroids in the newborn foal in the first days after birth. DHEA and...
Jacobson GA, Raidal S, Robson K, Narkowicz CK, Nichols DS, Haydn Walters E.Salbutamol is usually administered as a racemic mixture but little is known about the enantioselectivity of salbutamol pharmacokinetics in the lung. This study was designed to investigate enantiomer concentrations in lung tissue after inhaled dosing. Horses (n = 12) received racemic salbutamol 1000 μg via inhalation. Enantioselective ultra performance liquid chromatography-tandem mass spectrometry was used to determine salbutamol concentrations in pulmonary epithelial lining fluid (PELF) sampled 2, 5, 10 and 15 min after administration, in central lung (endoscopic bronchial biopsy) and per...
Satué K, Marcilla M, Medica P, Ferlazzo A, Fazio E.The objectives of this study were to establish reference values for serum concentrations of placental growth factor (PlGF) and haptoglobin (Hp), and to analyze whether the levels of oestrone sulphate (E1S) and progesterone (P4) are physiologically involved in the dynamic modifications of the above parameters in pregnant mares. A total of 30 healthy Spanish Purebred mares ranging in age 9.33 ± 3.31 years were studied during the 11 months of gestation. Serum concentrations of PlGF were detected by EIA, Hp using commercial Phase Haptoglobin assay and E1S and P4 levels through RIA. The serum ...
Schumacher SA, Yardley J, Bertone AL.Magnesium sulfate (MgSO ) was administered to calm competition horses. We evaluated the impact of regulatory requirements for the handling of blood samples on plasma ionized magnesium (iMg), ionized calcium (iCa), the iMg to iCa ratio, and pH. We hypothesized that iCa, iMg. and iMg/iCa would be similar among storage and collection methods. Four blood samples were collected from each of 50 horses on the same day: Group 1 - collection in a heparinized syringe and processed within hours in a clinical laboratory; Group 2 - collection into a plasma separator tube (PST) centrifuged just prior to ana...
Ellegren H, Andersson L, Johansson M, Sandberg K.A synthetic polynucleotide (TG)n was hybridized to equine DNA digested with HinfI and hypervariable hybridization patterns were obtained. Mendelian inheritance of these DNA fingerprinting patterns was confirmed by pedigree analysis. Estimates of the probabilities of identical band patterns in unrelated individuals of different breeds (Swedish Trotters, North Swedish Trotters, Thoroughbreds and Arabians) were in the range 1 x 10(-4) - 7 x 10(-6). The variability derived with the (TG)n probe in horses was higher than what we obtained with several other commonly used probes for DNA fingerprinting...
Minkwitz C, Schoon HA, Zhang Q, Schöniger S.Equine endometrosis, a frequent cause of subfertility, is characterized by periglandular fibrosis, and no treatment exists. Endometrial biopsies not only contain diseased glands, but also contain healthy glands and stroma. Myoepithelial (ME) and myofibroblastic (MF) markers are calponin, smooth muscle actin (SMA), desmin and glial fibrillary acidic protein (GFAP). Epithelial vimentin expression indicates epithelial to mesenchymal transition (EMT). The aim of this immunohistochemical study was to investigate whether biopsies with endometrosis express MF and ME markers and vimentin. Compared to ...
Martínez-Torrecuadrada JL, Díaz-Laviada M, Roy P, Sánchez C, Vela C, Sánchez-Vizcaíno JM, Casal JI.Fifteen horses were experimentally infected with African horse sickness virus (AHSV) serotype 4. To learn more about the time course of production and specificity of AHSV-specific antibodies, sera were analyzed by immunoblot analysis. Only animals that survived for more than 9 days were able to develop a humoral immune response detectable by immunoblotting. The earliest serological markers corresponded mainly to VP5, VP6, and NS2 and to a lesser extent to VP3, NS1, and NS3. Neutralizing antibodies to VP2 were not detected by immunoblotting, suggesting that they are mostly conformation dependen...
Boveland SD, Moore PA, Mysore J, Krunkosky TM, Dietrich UM, Jarrett C, Paige Carmichael K.Determine the effects of matrix metalloproteinases (MMPs)-2, -9, macrophage inflammatory protein-2 (MIP-2), tissue inhibitors of matrix metalloproteinase (TIMP)-1 and -2 by immunohistochemical expression in fungal affected and purulonecrotic corneas. Methods: Paraffin-embedded equine corneal samples; normal (n = 9), fungal affected (FA; n = 26), and purulonecrotic without fungi (PN; n = 41) were evaluated immunohistochemically for MMP-2, -9, MIP-2, TIMP-1 and -2. The number of immunoreactive inflammatory cells was counted and statistics analyzed. Western blot was performed to detect MMP-2, MMP...
Miszczak F, Shuck KM, Lu Z, Go YY, Zhang J, Sells S, Vabret A, Pronost S, Fortier G, Timoney PJ, Balasuriya UB.This study showed that under specifically defined conditions with respect to nucleic acid extraction method and testing reagents, a previously described real-time reverse transcription-PCR (rRT-PCR) assay (T1 assay) provides sensitivity equal to or higher than that of virus isolation for the detection of equine arteritis virus in semen.
Sun WC, Moore JN, Hurley DJ, Vandenplas ML, Linden JM, Murray TF.To evaluate anti-inflammatory effects of several novel adenosine receptor agonists and to determine their specificity for various adenosine receptor subtypes on neutrophils, cells heterologously expressing equine adenosine receptors, or equine brain membranes. Methods: Neutrophils isolated from 8 healthy horses. Methods: Radioligand binding experiments were performed to compare binding affinities of adenosine receptor agonists to equine adenosine A(1), A(2A), and A(3) receptor subtypes. Effects of these agonists on endotoxin-induced production of reactive oxygen species (ROS) by equine neutrop...
Loup B, André F, Avignon J, Lhuaire M, Delcourt V, Barnabé A, Garcia P, Popot MA, Bailly-Chouriberry L.Short half-life doping substances are, quickly eliminated and therefore difficult to control with traditional analytical chemistry methods. Indirect methods targeting biomarkers constitute an alternative to extend detection time frames in doping control analyses. Gene expression analysis (i.e., transcriptomics) has already shown interesting results in both humans and equines for erythropoietin (EPO), growth hormone (GH), and anabolic androgenic steroid (AAS) misuses. In humans, circulating cell-free microRNAs in plasma were described as new potential biomarkers for control of major doping agen...
Van Der Vekens N, Decloedt A, Sys S, Ven S, De Clercq D, van Loon G.Cardiac troponin I (cTnI) is a marker for detection of myocardial damage in horses. Many cTnI assays exist and medical studies have shown that the clinical performance of assays differs. The aim of this study was to compare two different cTnI assays in horses. Serum samples were taken from 23 healthy horses (group 1) and 72 horses with cardiac disease (group 2). Cardiac troponin I was determined using assay 1 in laboratory A (limit of detection, LOD, 0.03 ng/mL) and assay 2 in laboratories B and C (LOD 0.01 ng/mL). In group 1, a median cTnI concentration of <0.03 (<0.03-0.04) ng/mL and <0....
Russi RC, Garcia L, Cámara MS, Soutullo AR.Equine infectious anaemia (EIA) is controlled by the identification of seropositive animals. The official diagnostic method is the agar gel immunodiffusion (AGID) test, which detects antibodies against a viral core protein (p26). Although AGID is inexpensive and specific, the report of results takes considerable time and the test has low analytical sensitivity. Objective: To validate our in-house indirect ELISA , following the World Organization of Animal Health (OIE) criteria. Methods: Test validation. Methods: Synthetic peptides gp90 and gp45 were used as antigens in ELISA . Tests used for v...
Wong JKY, Choi TLS, Kwok KY, Lei ENY, Wan TSM.Equine hair is becoming an increasingly popular biological matrix for doping control of horse sports; one of the reasons for this is the significantly longer detection window hair can offer. Hair analysis opens up the opportunity for longitudinal monitoring of drug exposure which would otherwise not be possible with the more traditional and common biological matrices, such as urine and blood. As such, there is a need for more multi-target screening methods covering a broad range of prohibited substances in equine hair at the required sensitivities for equine doping control. This paper describe...
Cook VL, Holcombe SJ, Gandy JC, Corl CM, Sordillo LM.Monocytes are among the initial cells that interact with circulating LPS. Binding of LPS to monocyte surface receptors triggers an intracellular signaling cascade and results in the production of proinflammatory cytokines. Ethyl pyruvate, a stable derivative of pyruvate, has been effective in mitigating LPS induced alterations in isolated human monocytes. We hypothesized that ethyl pyruvate would suppress proinflammatory gene expression in LPS-stimulated equine monocytes without affecting cell viability. Equine monocytes were isolated from whole blood using a sediment-gradient centrifugation p...
White SD, Vandenabeele SI, Drazenovich NL, Foley JE.Malassezia-type yeasts previously have been observed on cytologic examination of the intermammary region of mares that presented with tail-head pruritus; topical antiyeast treatment resolved the pruritus. Further, Malassezia dermatitis has been observed in horses in intertriginous areas such as the udder and prepuce; the species of yeast was not confirmed. It is not known whether healthy mares or male horses can be carriers of this yeast in these body areas. Objective: Malassezia spp. are present in the intermammary region in healthy mares and the preputial fossa in healthy geldings. Methods: ...
Nishi S, Watabe H, Hirai H.The production of antibody to homologous alpha fetoprotein (AFP) in rabbits, rats, and horses by immunication with human AFP is reported. The antigens were administered subcutaneously 5 times at intervals of 7-10 days. Rabbits and dogs received 1 mg of human AFP/ml of the homologous pooled newborn serum with each injection while the rats received 1/2 of the dose. The horses received 5 mg/ml/injection. 2 weeks after the last injection, antisera were collected and immunologic assays were performed by the Ouchterlony method and the reversed version of the Mancini method. High titered antibodies w...
Hudson N, Mayhew I, Pearson G.Equine dysautonomia (grass sickness) is a frequently fatal disease of horses characterised by intestinal stasis. Interstitial cells of Cajal (ICC) are the pacemakers and mediators of neurotransmission in the gastrointestinal tract. Impaired ICC-mediated control of motility has been implicated in intestinal disorders in laboratory mammals, humans and in equine grass sickness. The aim of this study was to compare the in vitro electrical properties of ileum from grass sickness cases with horses free from gastrointestinal disease. Intracellular microelectrode recordings were made from smooth muscl...
Miño S, Kern A, Barrandeguy M, Parreño V.Group A rotaviruses (RVA) are important infectious agents associated with diarrhea in the young of several animal species including foals. Currently, a variety of diagnosis methods are commercially available, like ELISA, latex agglutination and immunochromatographic assays. These commercial tests are mainly designed for the detection of human RVA; its applicability in veterinary diagnosis has been poorly studied. The aim of this study was to compare the sensitivity and specificity of two commercial diagnostic kits, Pathfinder™ Rotavirus and FASTest Rota® strip, with an in-house KERI ELISA, ...
Ho EN, Wan TS, Wong AS, Lam KK, Stewart BD.Insulin administration can increase muscle glycogen by utilising hyperinsulinaemic clamps prior to sports events or during the recovery phases, and increase muscle size by its chalonic action to inhibit protein breakdown. In order to control insulin abuse in equine sports, a method to detect effectively the use of insulins in horses would be required. Besides the readily available human insulin and its synthetic analogues, structurally similar insulins from other species can also be used as doping agents. This study describes a method for the simultaneous detection of bovine, porcine and human...
