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.
Boone CW, Harell GS, Bond HE.Large-scale separation of mixtures of mammalian cells was obtained with the A-1X zonal centrifuge rotor and density gradients consisting of Ficoll dissolved in modified Eagle's MEM suspension-culture medium. The cells remained viable as tested by plating efficiency or by motility observed with time-lapse photography. Rabbit thymocyte and HeLa cell mixtures were separated with 99 and 89 per cent purity, respectively. Mixtures of thymocytes and suspension-cultured, human acute leukemia cells (Roswell Park strain LKID) were separated with 93 and 91% purity, respectively. HeLa cells were isolated ...
Hampil B, Melnick JL.This paper summarizes the results of the second part of co-operative studies undertaken by the WHO International Reference Centre for Enteroviruses and a number of WHO Regional Virus Reference Centres, WHO Virus Collaborating Laboratories and other laboratories in a comprehensive testing programme of enterovirus equine antisera. The studies were designed to appraise the specificity of immune serum prepared in horses against 16 representative prototype enteroviruses (polioviruses 2 and 3, coxsackieviruses A7, B1, B2, B4, B5 and B6 and echoviruses 1, 2, 3, 5, 7, 9, 12 and 17). Tests for neutrali...
Dhakal P, Tsunoda N, Nambo Y, Taniyama H, Nagaoka K, Watanabe G, Taya K.Although equine gestation is unique from the standpoint of fetal gonadal enlargement and regression, the activator of this process is still unknown. The present study aimed to show a possible role of activin during equine gestation. In the first experiment, weekly plasma samples from six pregnant mares were used to measure activin A. In the second experiment, eight pregnant mares carrying female (gestational days 110, 140, 180, and 270) and male fetuses (gestational days 120, 180, 225, and 314) were used for immunohistochemistry of activin receptors (IA, IB, IIA, IIB), and their intracellular ...
Van Beek H, Baars AJ.Metallothioneins (MTs) were separated and quantitated by reversed-phase high-performance liquid chromatography (RP-HPLC), in direct combination with atomic absorption spectrometry (AAS) for quantitation of the metal contents in MTs. MTs were eluted from an RP-8 column with a gradient of Tris buffer pH 7.0 and methanol, and were detected by UV absorbance (220 nm). Commercially available purified MTs from horse kidney and rabbit liver were analyzed for purity and metal composition. One lot of horse kidney yielded only 50% of the estimated value. In some cases, the certified metal content differe...
de Val N, Herschbach H, Potier N, Dorsselaer AV, Crichton RR.An essential difference between eukaryotic ferritins and bacterioferritins is that the latter contain naturally, in vivo haem as Fe-protoporphyrin IX. This haem is located in a hydrophobic pocket along the 2-fold symmetry axes and is liganded by two Met 52. However, in in vivo studies, a cofactor has been isolated in horse spleen apoferritin similar to protoporphyrin IX; in in vitro experiments, it has been shown that horse spleen apoferritin is able to interact with haem. Studies of haemin (Fe(III)-PPIX) incorporation into horse spleen apoferritin have been carried out, which show that the me...
Bossart KN, Wang LF, Eaton BT, Broder CC.Hendra virus (HeV) is an emerging paramyxovirus first isolated from cases of severe respiratory disease that fatally affected both horses and humans. Understanding the mechanisms of host cell infection and cross-species transmission is an important step in addressing the risk posed by such emerging pathogens. We have initiated studies to characterize the biological properties of the HeV envelope glycoproteins. Recombinant vaccinia viruses encoding the HeV F and G open reading frames were generated and glycoprotein expression was verified by metabolic labeling and detection using specific antis...
Wilkins PA.Rapid evaluation and intervention is a requirement and a characteristic of patient management in neonatal intensive care units, and this applies for equine neonates also. Appropriate interventions are based on solid knowledge of age, maturity, and species-specific differences in reference ranges. Point-of-care (POC) testing devices speedup decision making regarding treatments and interventions. However, there are potential limitations of these devices when applied to age groups and species beyond those they were specifically developed for. This article discusses the age-specific differences in...
Bougrine SI, Fihri OF, Fehri MM.A Western immunoblotting procedure has been developed for the detection of African horse sickness virus (AHSV) protein-specific antibody responses. This assay readily identifies antibodies specific for at least 4 distinct, AHSV proteins, including VP5, NS1, NS2 and NS3/NS3a. By using the AHSV non-structural proteins as 'markers', the Western blotting procedure could be employed to provide a reliable means of discriminating between animals vaccinated with a purified, inactivated AHSV vaccine and those either naturally infected or vaccinated with a live, attenuated AHSV vaccine.
Thougaard AV, Jaliashvili I, Christiansen M.The glycoprotein tetranectin (TN) found in human serum is a 90-kDa homotrimeric C-type lectin binding Ca2+, heparin and plasminogen kringle 4. TN is suggested as being implicated in tissue remodelling. The antigenic reactivity of putative TN was examined in serum from 14 different animal species using three sandwich enzyme immunoassays for human TN. Crab-eating macaque serum showed the strongest reaction, followed by horse and cat. Serum from cow, goat, pig, mouse and chicken reacted weakly, while dog, trout, and the amphibian and the reptile species did not react. The TN-like protein from mac...
Lorenzo PL, Liu IK, Illera JC, Picazo RA, Carneiro GF, Illera MJ, Conley AJ, Enders AC, Illera M.Epidermal growth factor (EGF) has been reported to promote different functions in mammalian ovaries, including oocyte maturation. The aim of the present study was to establish: that EGF influences oocyte maturation in ovine and equine, that a tyrosine kinase-dependent intracellular mechanism mediates EGF effect and, that EGF-R receptor is detectable in ovarian follicles by immunohistochemistry methods. Selected ovine and equine oocytes were aspirated from 2-5 mm (ovine) or 25 mm (equine) follicles and cultured in TCM 199 for 22 (ovine) or 36 hours (equine). They are then subjected to culture w...
Rice DW, Blake CC.Crystals of horse muscle 3-phosphoglycerate kinase have been grown in the presence of a wide variety of substrates using either potassium tartrate or polyethylene glycol as a precipitant. In those grown from polyethylene glycol, two related crystal forms have been obtained by varying the nature of the substrates present in the crystallization medium. In order to obtain one of these forms, form B, the presence of the substrate 3-phosphoglycerate appears to be essential. The two crystal forms are not interconvertible by simple diffusion experiments and the crystals grown in the absence of 3-phos...
Broström H, Obel N, Perlmann P.Human lymphocytes displayed a frequent natural cytotoxicity (NK) in vitro against normal equine dermal fibroblasts (ED) and against equine tumour cells of a virus-containing cell line (Mc-1). Similarly, human normal sera contained antibodies that induced antibody-dependent cellular cytotoxicity (ADCC) by normal human lymphocytes against the same target cells. Both NK and ADCC varied for different donors. For individual donors, however, cytotoxicity against the two target cells was significantly correlated both in NK and ADCC. For ED there was also a significant correlation between ADCC and NK ...
Archambault D, St-Louis MC, Martin S.The genome of equine arteritis virus (EAV) produces a 3' coterminal-nested set of six subgenomic (sg) viral RNAs during virus replication cycle, and each set possesses a common leader sequence of 206 nucleotides (nt) in length derived from the 5' end of the viral genome. Given the presence of the leader region within both genomic and sg mRNAs, it is likely to contain cis-acting signals that may interact with cellular or viral proteins for RNA synthesis. Gel mobility shift assays indicated that proteins in Vero cell cytoplasmic extracts formed complexes with the positive (+) and negative (-) st...
Neuwirth L, Romine C.The construction of ancillary equipment used to improve image quality and reduce personnel radiation exposure in the equine nuclear medicine laboratory is illustrated. The devices include a self supporting lead sheet for shielding the distal limb or limb pair, a hanging lead sheet for shielding the proximal limb, a lead square for shielding the urinary bladder or jugular catheter, a restraining board for acquiring a palmar view of the foot, a head support to stabilize the head for imaging and a head support for stabilizing the neck for imaging. The restraining board and head supports decrease ...
Thomas RJ.An antigen for the gel diffusion test for equine infectious anaemia (EIA) was prepared from the spleen of a horse experimentally infected with the CQ strain of the virus. The antigen produced a single, distinct line of precipitation when tested against a range of known positive serums, and did not react with pre-inoculation and known negative serums. Extracts prepared from uninfected spleens displayed no reaction when similarly tested. Serum from 34 of 451 Queensland horses contained detectable levels of antibody to EIA virus. The positive serums were from horses in widely separated areas of t...
Alben JO, Bare GH.Infrared absorption spectra of the alpha-104 (G11) cysteine SH group have been observed for aqueous solutions of hemoglobin derivatives from humans, pigs, and horses. The center frequencies ((nu)SH) show ligand sensitive patterns that are similar for the three species, with (nu)SH (HbCO) <(nu)SH (HbO(2) ~ HbCN) < (nu)SH (Hb(+)) <<(nu)SH (deoxyHb) for human and pig hemoglobins. The alpha-104 SH group is most strongly H-bonded (smallest (nu)SH), has the greatest range of (nu)SH (Hb ? HbCO) in human hemoglobin, and is least strongly H-bonded and has the smallest range of (nu)SH (Hb ? HbCO) in hor...
Hewetson M, Aaltonen K, Tulamo RM, Sankari S.A simple and accurate method for quantifying sucrose in equine serum that can be applied to sucrose permeability testing in the horse was developed and validated using gas chromatography with flame ionization detection. The assay provided an acceptable degree of linearity, accuracy, and precision at concentrations of sucrose as low as 2.34 μmol/l and as high as 20.45 μmol/l. Percentage recovery of sucrose from serum ranged from 89% to 102%; repeatability and intermediate precision (relative standard deviation) ranged from 3.6% to 6.7% and 4.1% to 9.3%, respectively. The limit of detection wa...
Woods WE, Chay S, Houston T, Blake JW, Tobin T.Interference or 'masking' in thin layer chromatography occurs when the presence of one drug on a thin layer plate physically obscures or interferes with the detection of another drug. We investigated the ability of phenylbutazone and oxyphenbutazone to mask or interfere with the detection by high performance thin layer chromatography (HPTLC) of basic drugs used illegally in horse racing. Of fifty-five basic drugs called 'positive' since 1981 by laboratories affiliated with the Association of Official Racing Chemists (AORC), forty did not comigrate with phenylbutazone or oxyphenbutazone and cou...
Cutler C, White DL, Viljanto M.SR-9009 is a synthetic compound widely available to purchase online as 'supplement' products due to its potential performance-enhancing effects, presenting a significant threat with regard to doping control in sport. In vitro metabolism with equine liver microsomes was performed to identify potential targets for detection of SR-9009. Six metabolites were identified, with the most abundant consisting of N-dealkylated metabolites (M1-M3). The addition of the identified metabolites to high-resolution accurate mass databases resulted in a positive finding for the N-dealkylated metabolite M1 of SR-...
Hoogewijs M, Rijsselaere T, De Vliegher S, Vanhaesebrouck E, De Schauwer C, Govaere J, Thys M, Hoflack G, Van Soom A, de Kruif A.Three experiments were conducted to evaluate the impact of centrifugation on cooled and frozen preservation of equine semen. A standard centrifugation protocol (600 x g for 10 min=CP1) was compared to four protocols with increasing g-force and decreased time period (600 x g, 1200 x g, 1800 x g and 2400 x g for 5 min for CP2, 3, 4, and 5, respectively) and to an uncentrifuged negative control. In experiment 1, the influence of the different CPs on sperm loss was evaluated by calculating the total number of sperm cells in 90% of the supernatant. Moreover, the effect on semen quality following ce...
Xu J, Cai J, Suresh M, Peek SF, Darien BJ.Equine PSGL-1 (ePSGL-1) is widely expressed on equine PBMC as a homodimer with sialylation (sLeX) modifications that contribute to P-selectin binding affinity. To investigate the role of other potential post-translational modifications required for high-affinity P-selectin binding, ePSGL-1 was transfected into CHO cells expressing equine FucT-VII and/or C2GnT. P-selectin-IgG chimera binding by ePSGL-1 transfected into CHO cells only occurred when both FucT-VII and C2GnT were expressed, establishing that fucosylation and core-2 branching are required as post-translational modifications for high...
Dell'aquila ME, Fusco S, Lacalandra GM, Maritato F.The aim of this study was to develope an efficient and reproducible procedure for in vitro maturation (IVM) and fertilization (IVF) in the horse. Cumulus-oocyte complexes (COCs) recovered from the ovaries of mares slaughtered during the breeding season were morphologically evaluated, and those showing a compact cumulus and homogeneously appearing cytoplasm were selected for culture. Effects on the maturation of estrous mare serum (EMS) versus estrous cow serum (ECS) as medium supplement were also evaluated (Experiment 1). In Experiment 2, the fertilization of in vitro matured oocytes with froz...
Poole AW, Heath MF, Evans RJ.Pre-incubation of equine platelets in platelet-rich plasma with adenosine 5'-diphosphate (ADP) induced a reduction in aggregation responsiveness to subsequent additions of ADP. The desensitisation was shown to be homologous since the responsiveness to platelet-activating factor, thrombin, collagen, 5-hydroxytryptamine or ionomycin remained unchanged. Adenosine 5'-(beta-thio)-diphosphate (ADP beta S), a non-hydrolysable analogue of ADP, was shown to act as an agonist inducing aggregation by interaction with the ADP receptor. ADP beta S was then used in the desensitisation studies in which resid...
Johannessen MK, Skretting G, Ytrehus B, Røed KH.Endochondral bone formation is an important process in development and growth of the skeleton; still many of the mechanisms of growth cartilage remain unknown. The aim of this study was to identify genes specifically expressed in growth cartilage by constructing a subtraction cDNA library of the articular-epiphyseal cartilage complex from neonatal foal. Two hundred and eighty-four differently expressed clones, representing five novel and 37 known genes, were detected by subtraction hybridization. The tissue specificity of these genes was verified by reverse Northern analysis, and tissue distri...
Slusarewicz P, Pagano S, Mills C, Popa G, Chow KM, Mendenhall M, Rodgers DW, Nielsen MK.Intestinal parasites are a concern in veterinary medicine worldwide and for human health in the developing world. Infections are identified by microscopic visualisation of parasite eggs in faeces, which is time-consuming, requires technical expertise and is impractical for use on-site. For these reasons, recommendations for parasite surveillance are not widely adopted and parasite control is based on administration of rote prophylactic treatments with anthelmintic drugs. This approach is known to promote anthelmintic resistance, so there is a pronounced need for a convenient egg counting assay...
Smith ML, Chapman CB.An acepromazine (ACP) hapten was synthesised, coupled to bovine serum albumin and injected into a horse to produce antibodies to the drug. A competitive ELISA was developed whereby ACP attached to the solid phase via lysozyme competed with free ACP present in phosphate buffered saline, horse serum or horse urine for limiting amounts of antibody. The assay could detect the presence of ACP and, or, some of its metabolites in horse urine for at least 25 hours after intravenous injection of 0.1 mg kg-1 ACP maleate, but because of non-specific interference, horse serum could not be used. As little ...
Pasing SS, Aurich C, von Lewinski M, Wulf M, Krüger M, Aurich JE.An important factor influencing stallion fertility is the microbial contamination of semen. Aims of this study were to investigate changes in the microbiological population of the genital mucosa and semen in artificial insemination stallions (n=16) from before to after one breeding season (February-August). MALDI-TOF-MS (matrix assisted laser desorption/ionization time-of-flight mass spectrometry) was used for identification of microbial agents. For bacteriology, swabs from the urethral opening, urethral fossa and penile sheath as well as semen were collected at 4-week-intervals. For semen mot...
Sampaio BFB, Nogueira BG, Souza MIL, Silva EVDCE, Zúccari CESN.The aim of this experiment was to evaluate the effects of adding ascorbic acid 2-glucoside (AA2G), a water-soluble antioxidant and stable derivative of ascorbate, to the semen extender and compare it to the addition of vitamin C (Vit. C) and the fat-soluble antioxidant α-tocopherol (α-Toh), both individually and in combination, on the seminal variables of equine sperm submitted to cooling for 72 h. We used two ejaculates from 10 stallions and evaluated them for motility, membrane integrity, chromatin fragmentation, mitochondrial activity and lipid peroxidation. In the analysis of lipid pero...
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.
Magnarelli LA, Ijdo JW, Van Andel AE, Wu C, Oliver JH, Fikrig E.To test serum samples of dogs and horses by use of class-specific recombinant-based ELISA for establishing a diagnosis of granulocytic ehrlichiosis attributable to infection with organisms from the Ehrlichia phagocytophila genogroup. Methods: Serum samples from 43 client-owned dogs and 131 horses (81 with signs of acute illness and 50 without signs of disease). Methods: Serum samples were analyzed, using ELISA with a recombinant 44-kd protein antigen for IgM and IgG antibodies to the human granulocytic ehrlichiosis (HGE) agent (NCH-1 strain). Western blot analyses, using infected human promyel...
Wise LM, Bodaan CJ, Mercer AA, Riley CB, Theoret CL.Wounds in horses often exhibit sustained inflammation and inefficient vascularization, leading to excessive fibrosis and clinical complications such as "proud flesh". Orf virus-derived proteins, vascular endothelial growth factor (VEGF)-E and interleukin (ovIL)-10, enhance angiogenesis and control inflammation and fibrosis in skin wounds of laboratory animals. Objective: The study aimed to determine if equine dermal cells respond to VEGF-E and ovIL-10. Equine dermal cells are expected to express VEGF and IL-10 receptors, so viral protein treatment is likely to alter cellular gene expression an...
Horká M, Kubíček O, Kubesová A, Rosenbergová K, Kubíčková Z, Šlais K.Influenza A is viral disease, which is a cause of yearly epidemics and, potentially, pandemics. The conventional techniques used today are equipment-demanding, time-consuming and laborious. Recently, we have confirmed that the capillary isoelectric focusing is a suitable fast alternative for the verifying of virus purity. In the wide pH gradient of pH range 2.0-7.5 the isoelectric points for subtypes of equine (H3N8) and swine (H1N2) influenza A viruses were determined approximately as 6.6 and 6.5, respectively. In this contribution we have verified these findings using different isolates of d...
