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Topic:Laboratory Methods
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.
Construction of a stable non-mucoid deletion mutant of the Streptococcus equi Pinnacle vaccine strain. Streptococcus equi causes equine strangles, a purulent lymphadenopathy of the head and neck. An avirulent, non-encapsulated strain (Pinnacle) has been used widely in North America as an intranasal vaccine. The aim of the study was to create a specific mutation of the hyaluronate synthase (hasA) gene in Pinnacle to permanently abolish the production of capsule and provide an easily recognisable genetic marker. An internal fragment of hasA was generated by PCR and cloned into pTW100 (Microscience, UK). An encapsulated revertant of Pinnacle was then transformed with the recombinant plasmid by ele...
Competition studies in horse spleen ferritin probed by a kinetically inert inhibitor, [Cr(TREN)(H(2)O)(OH)](2+), and a highly luminescent Tb(III) reagent. The ability of ferritin as an Fe(II) detoxifier and Fe(III) storage protein is limited by its ability to recognize and incorporate Fe(II), which is then oxidized and mineralized at internal protein sites. The Cr(III) amine complex [Cr(N(CH(2)CH(2)NH(2))(3)(H(2)O)(OH)](2+) [abbreviated as Cr(TREN)] is a kinetically inert inhibitor of iron incorporation and mineralization in ferritin. Unlike other inhibitors, Cr(TREN) can only exchange its two aqua/hydroxy ligands. Competition studies between Cr(TREN) and Tb(III) binding have been performed in horse spleen ferritin (HoSF) to probe uptake of Fe(I...
Surface plasmon resonance measurement of pH-induced responses of immobilized biomolecules: conformational change or electrostatic interaction effects? Recently, the observation of pH-induced conformational changes of biomolecules supported on carboxymethyldextran (CMD)-coated surfaces measured using surface plasmon resonance (SPR) has been reported. However, it is apparent that the evidence reported in the literature is ambiguous. The research presented in this paper describes investigations to study the changing SPR signal of immobilized biomolecules as a function of varying pH, to provide a detailed understanding of the origin of the pH-induced changes in the SPR profile. SPR measurements were performed with cytochrome c, concanavalin A, a...
Kinetic barriers to the folding of horse cytochrome C in the reduced state. To determine the kinetic barrier in the folding of horse cytochrome c, a CO-liganded derivative of cytochrome c, called carbonmonoxycytochrome c, has been prepared by exploiting the thermodynamic reversibility of ferrocytochrome c unfolding induced by guanidinium hydrochloride (GdnHCl), pH 7. The CO binding properties of unfolded ferrocytochrome c, studied by 13C NMR and optical spectroscopy, are remarkably similar to those of native myoglobin and isolated chains of human hemoglobin. Equilibrium unfolding transitions of ferrocytochrome c in the presence and the absence of CO observed by both e...
Lipase activity in stallion seminal plasma and the effect of lipase on stallion spermatozoa during storage at 5 degrees C. Previous studies have demonstrated a detrimental effect of seminal plasma on the maintenance of motility of cooled equine spermatozoa; however, the mechanism for the adverse effect of seminal plasma during cooled storage remains undetermined. In goats, a glycoprotein component of bulbourethral gland secretion contains lipase activity that is detrimental to sperm motility when stored in skim milk-based extenders. The objective of the current study was to determine the amount of lipase activity in stallion seminal plasma and to determine the effect of added lipase on spermatozoal motility during...
Cytogenetic localization of 136 genes in the horse: comparative mapping with the human genome. The aim of this study was to increase the number of type I markers on the horse cytogenetic map and to improve comparison with maps of other species, thus facilitating positional candidate cloning studies. BAC clones from two different sources were FISH mapped: homologous horse BAC clones selected from our newly extended BAC library using consensus primer sequences and heterologous goat BAC clones. We report the localization of 136 genes on the horse cytogenetic map, almost doubling the number of cytogenetically mapped genes with 48 localizations from horse BAC clones and 88 from goat BAC clon...
Immunohistochemical localization of chromogranin a in the acinar cells of equine salivary glands contrasts with rodent glands. We investigated the existence of chromogranin A (CgA) in salivary glands of the horse by Western blotting and enzyme immunoassay (EIA) using an antiserum against a peptide sequence of equine CgA. We also compared its cellular distribution between the horse and rat salivary glands with a tyramide signal amplification immunofluorescence technique. Western blotting gave three significant immunoreactive bands (74, 56 and 48 kDa) in adrenal medulla and three major salivary glands of horses. Immunoreactivities for CgA measured by EIA in horses were 154.05 +/- 41.46, 20.32 +/- 5.59 and 4.43 +/- 2.23 ...
Different contractile effects of alpha1- and alpha2-adrenergic agonists on horse isolated common digital artery smooth muscle ring preparations in vitro. Despite assays on ring preparations in vitro confirmed that the vasoconstrictor sympathetic control in the horse common digital artery mainly depends on alpha(1)-adrenoceptors stimulation, selective alpha(2)-adrenoceptor agonists were investigated under the same experimental conditions. Both detomidine (DET) and UK 14304 differed from noradrenaline (NA) and phenylephrine (PHE) in provoking contractile effects which were slowly onsetting, concentrations-unrelated and unremovable by repeated washings. While prazosin (PRA) clearly antagonized the effects of NA and PHE, neither pre- nor post-treat...
Reclassification of equine isolates previously reported as Actinobacillus equuli, variants of A. equuli, Actinobacillus suis or Bisgaard taxon 11 and proposal of A. equuli subsp. equuli subsp. nov. and A. equuli subsp. haemolyticus subsp. nov. Members of Bisgaard taxon 11 have been isolated from horses. These bacteria are of importance in the veterinary clinic and also to the medical profession, since they may be isolated from infected wounds of humans bitten by horses. Six strains from different continents were identified as taxon 11, with 16S rRNA similarities between 98.0 and 99.7%. A single isolate that represented the so-called (+)L-arabinose-positive Actinobacillus equuli isolated from a diseased foal showed 99.9% 16S rRNA similarity to the type strain of A. equuli. DNA-DNA hybridizations showed that (+)L-arabinose-positive st...
Assignment of the horse grey coat colour gene to ECA25 using whole genome scanning. The dominant grey coat colour gene of horses has been mapped using a whole genome scanning approach. Samples from a large half-sibling pedigree of Thoroughbred horses were utilized in order to map the grey coat colour locus, G. Multiplex groups of microsatellite markers were developed and used to efficiently screen the horse genome at a resolution of approximately 22 cM, based on an estimated map length for the horse genome of 2720 cM. The grey gene was assigned to chromosome 25 (ECA25), one of the smaller acrocentric horse chromosomes. Based on the current state of knowledge of conserved synt...
Nucleation rate determination by a concentration pulse technique: application on ferritin crystals to show the effect of surface treatment of a substrate. The nucleation of horse spleen ferritin (HSF) crystals on substrates was investigated using a new modification of the double pulse technique. The influence of three different structureless substrates (glass, glass covered by methyl groups and poly-L-lysin template) on the nucleation was studied. The boundaries in the phase-diagram, which separate zones of crystal nucleation and growth were obtained by keeping pH = 5.0, and using CdSO(4) as crystallizing agent. The steady-state nucleation rates were determined. The energy required for critical nuclei formation was evaluated (10(-13) erg) and th...
Correlation between the osmotic second virial coefficient and solubility for equine serum albumin and ovalbumin. The Haas - Drenth - Wilson (HDW) (Haas et al., 1999) theoretical model was used to correlate osmotic second virial coefficient (B) values with solubility (S) values for equine serum albumin (ESA) and ovalbumin for corresponding solution conditions. The best fit from the theoretical model was compared to experimental S versus B data. B values were experimentally measured using static light scattering. Solubilities of ESA were estimated using a sitting drop method. When the experimental data for S versus B were plotted, an excellent fit for ESA was obtained according to the HDW model. The result...
Presence of in vitro electrical activity in the ileum of horses with enteric nervous system pathology: equine dysautonomia (grass sickness). 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...
Evaluation of the SNAP foal IgG test for the semiquantitative measurement of immunoglobulin G in foals. The SNAP Foal IgG test (IDEXX) as evaluated for its accuracy and usefulness by measuring blood samples collected from 42 foals between 24 and 48 hours after birth. The results were compared with the single radial immunodiffusion (SRID) test as the reference method. The SNAP test was quick and easy to perform, and the results were similar to those obtained by SRID in 64 per cent of the samples. The best results were found with low ( 800 mg/dl) concentrations of immunoglobulin G, with an accuracy of 80 per cent and 89 per cent, respectively. The intermediate concentrations were usually lower whe...
Selection of recently isolated colicinogenic Escherichia coli strains inhibitory to Escherichia coli O157:H7. Escherichia coli strains were screened for their ability to inhibit E. coli O157:H7. An initial evaluation of 18 strains carrying previously characterized colicins determined that only colicin E7 inhibited all of the E. coli O157:H7 strains tested. A total of 540 strains that had recently been isolated from humans and nine different animal species (cats, cattle, chickens, deer, dogs, ducks, horses, pigs, and sheep) were tested by a flip-plating technique. Approximately 38% of these strains were found to inhibit noncolicinogenic E. coli K12 strains. The percentage of potentially colicinogenic E...
Measurement of leptin and insulin-like growth factor-I in seminal plasma from different species. The multi-functional proteins, insulin-like growth factor-I (IGF-I) and leptin were present in seminal plasma from different species. Concentrations of IGF-I in equine and porcine semen were 20 and 17.5 ng/ml, respectively. Seminal plasma concentrations of leptin were 1 ng/ml in human and 11 ng/ml in porcine samples.
Detection of Salmonella spp in fecal specimens by use of real-time polymerase chain reaction assay. To use real-time polymerase chain reaction (PCR) technology to develop a highly sensitive and specific diagnostic assay for the detection of Salmonella spp in fecal specimens. Methods: 299 fecal specimens from cattle, horses, and dogs. Methods: Enrichment of fecal specimens was followed by genomic DNA extraction by use of commercially available isolation kits. Real-time PCR assay was performed to target a Salmonella spp-specific DNA segment. Results of real-time PCR assay were compared with bacterial culture results to determine relative sensitivity and specificity. Results: Use of the spaQ pr...
Agalactia in mares fed with grain contaminated with Claviceps purpurea. This article reports an outbreak of intoxication of female horses with Claviceps purpurea in southern Brazil. The outbreak affected twelve pregnant mares which were fed with black oat (Avena strigosa) during the pre-delivery period. Underdevelopment of the mammary gland in the pre-delivery period resulting in post-delivery agalactia was the most pronounced finding. These mares delivered weak and unviable foals, which showed no suckling reflex and died within a few hours of birth. Laboratory analysis of oat samples fed to the animals resulted in the identification of Claviceps purpurea scleroti...
Capacitation-like changes in equine spermatozoa throughout the cryopreservation process. Chlortetracycline (CTC) fluorescence staining analysis was used to investigate cryopreservation-induced capacitation-like changes in equine spermatozoa. Freshly ejaculated spermatozoa were found to display a high proportion of F pattern cells (uncapacitated; 93.6%) and a lower proportion of B pattern (capacitated; 5.4%) and AR pattern (acrosome-reacted; 1%) cells. Following cryopreservation in modified Kenney's medium, capacitation-like changes were observed. There was a significant increase in the proportion of spermatozoa displaying the B pattern (64.8%; P<0.001) and AR pattern (32.8%; P&...
Quantitative detection of salmeterol after inhalation in equine urine by liquid chromatography/tandem mass spectrometry. A sensitive, accurate and precise liquid chromatography/tandem mass spectrometry (LC/MS(2)) method was developed for the quantification of salmeterol in the urine of horses. The method consists of a liquid-liquid extraction with tert-butylmethyl ether and isopropanol at pH 12 after enzymatic hydrolysis. The extracts are analysed using an LC/MS system equipped with an electrospray ionisation (ESI) probe. Method validation showed excellent linearity, specificity, accuracy, precision and intra-laboratory repeatability and reproducibility. The limit of quantitative detection was 0.25 ng/mL and the...
A rapid and simple method for the separation of pure lymphocytes from horse blood. A method for the separation of pure and viable lymphocytes and granulocytes from the same blood sample in horses was reported. By centrifuging equine heparinized blood at 100 xg for 10 min at room temperature (r.t.), the resulting supernatant plasma was an almost pure (97.71 +/- 0.30%; n = 15) suspension of highly viable (98.72 +/- 0.28%) lymphocytes. When sodium citrate was used as an anticoagulant, lymphocyte suspensions collected in the same manner showed lower purity (87.89 +/- 1.59%; n = 9) and higher yields (56.56 +/- 3.89%, n = 9 versus 36.11 +/- 2.23%, n = 15). Where needed, a further ...
Quantification of clenbuterol in equine plasma, urine and tissue by liquid chromatography coupled on-line with quadrupole time-of-flight mass spectrometry. Clenbuterol (CBL) is a potent beta(2)-adrenoceptor agonist used for the management of respiratory disorders in the horse. The detection and quantification of CBL can pose a problem due to its potency, the relatively low dose administered to the horse, its slow clearance and low plasma concentrations. Thus, a sensitive method for the quantification and confirmation of CBL in racehorses is required to study its distribution and elimination. A sensitive and fast method was developed for quantification and confirmation of the presence of CBL in equine plasma, urine and tissue samples. The method i...
Method for preparing thin sections of untreated equine hoof horn for electron microscopic examination. The preparation of hard tissues such as the equine hoof horn for electron microscopic examination is very difficult. In particular the penetration of fixatives and chemicals used during fixation and embedding is a problem. The objective of this study was to find and implement an alternative method enabling the preparation of high-quality thin sections of hoof horn and other hard tissue, which maintains the hard tissue ultrastructure and can be used for immuno-labeling. Compared to commonly used fixation and embedding techniques, the preparation of thin sections from untreated material method s...
Spectroscopic and electrochemical studies of horse myoglobin in dimethyl sulfoxide. This paper reports the first report of rapid, reversible direct electron transfer between a redox protein, specifically, horse myoglobin, and a solid electrode substrate in nonaqueous media and the spectroscopic (UV-vis, fluorescence, and resonance Raman) characterization of the relevant redox forms of myoglobin (Mb) in dimethyl sulfoxide (DMSO). In DMSO, the heme active site of metmyoglobin (metMb) appears to remain six-coordinate high-spin, binding water weakly. Changes in the UV-fluorescence spectra for metMb in DMSO indicate that the protein secondary structure has been perturbed and sugge...
An activator protein-1 complex mediates epidermal growth factor regulation of equine glycoprotein alpha subunit expression in trophoblast cells. Equids and primates are the only species known to express the placental hormone chorionic gonadotropin (CG). CG is a member of the heterodimeric glycoprotein family and is composed of an alpha subunit linked to a hormone-specific beta subunit. Previously, we have reported that epidermal growth factor (EGF) regulates the equine glycoprotein hormone alpha subunit promoter through a protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) signal transduction pathway in trophoblasts. The current study investigates the regulatory element/factors involved in the induction of equine glycoprotei...
Development of a multiplex real-time reverse transcriptase-polymerase chain reaction for equine infectious anemia virus (EIAV). A single-tube reverse transcriptase-polymerase chain reaction (RT-PCR) using a fluorogenic real-time PCR detection method is described for the quantitation of equine infectious anemia virus (EIAV) RNA in the plasma of equids. To compensate for variations inherent in sample preparation a multiplex real-time RT-PCR system was developed that permitted the simultaneous calculation of the nucleic acid recovery rate along with the copy number of viral RNA molecules. Detection of EIAV RNA was linear from 10(9) to 10(1) molecules with intra- and inter-assay variability of less than 1% at 10(8), 10(6),...
Evaluation of microbial culture techniques for the isolation of Pythium insidiosum from equine tissues. The purpose of this study was to evaluate the effects of sample handling, storage, and culture techniques on the isolation of Pythium insidiosum from infected equine tissues. Tissue and kunker samples obtained immediately posteuthanasia from a horse with subcutaneous pythiosis were used to assess the effects of sample type (kunkers vs. tissues), media type (selective vs. nonselective), storage technique, and storage time on P. insidiosum isolation rate. Overall, isolation rates were higher from fresh kunkers (94.6%) and stored kunkers (76.4%) than from fresh tissues (8.3%) or stored tissues (4...
Effect of extraction time and acid concentration on the separation of proglycogen and macroglycogen in horse muscle samples. The objective of this study was to determine whether the concentrations of proglycogen (PG) and macroglycogen (MG) in biopsy samples of horse muscle are influenced by extraction time or perchloric acid (PCA) concentration. In study 1, individual muscle-biopsy samples from 10 horses were divided into 4 parts each and then randomly subjected to 4 periods of extraction (10, 20, 60, or 120 min) with 1.5 M PCA. In study 2, individual muscle-biopsy samples from 6 horses were divided into 24 pieces each and then randomly subjected to 12 combinations of extraction time (10, 20, 30, or 40 min) and PCA ...
Flow cytometry: clinical applications in equine medicine. The use of flow cytometry in veterinary diagnostics is becoming a valuable clinical tool with a broad range of applications. Physical characteristics of cells can be determined by the flow cytometer laser and electronics through the measurement of changes in light scatter properties. Other components and functions of cells can be defined through the application of fluorochrome dyes that have an affinity for cellular components. Traditionally, common clinical applications are immunophenotyping of cells of the hematopoietic system with fluorescent-labeled antibodies raised against specific cell ...
A PCR-RFLP for KIT associated with tobiano spotting pattern in horses. An MspI polymorphism was identified in intron 13 of the equine homologue of proto-oncogene c-kit (KIT) by comparing DNA sequences from horses with solid coat colour and horses homozygous for the tobiano spotting (To) gene. The allele associated with solid coat colour was designated KM0, while the allele associated with the tobiano pattern created an additional MspI restriction site and was designated KM1. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) studies using DNA from hair follicles demonstrated that all 129 of 129 tobiano patterned horses possessed the KM1...
