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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.
Permeation of small molecules into the cavity of ferritin as revealed by proton nuclear magnetic resonance relaxation. The NMR relaxation technique was used to investigate the permeation of molecules into the cavity of ferritin. Spin-lattice relaxation times in the rotating frame of various probe molecules were measured for solutions of recombinant horse L-apoferritin without iron and horse spleen apoferritin with very small amounts of ferric ions. The results show that molecules larger than the size of the ferritin channels can pass through the channels into the ferritin interior, and that the maximum size of molecules for the permeation is smaller than maltotriose.
Proteoglycan metabolism of equine articular cartilage and its modulation by insulin-like growth factors. The effect of human recombinant insulin-like growth factor 1 (rhIGF-1) on proteoglycan (PG) metabolism of full thickness equine articular cartilage explants was investigated. PG synthesis was stimulated at all ages, but higher concentrations of rhIGF-1 were required for maximal stimulation of adult cartilage. There were no changes in the hydrodynamic size, electrophoretic heterogeneity or composition of proteoglycans isolated from rhIGF-1-stimulated cartilage. rhIGF-1 reduced the rate of turnover of both newly synthesized and endogenous proteoglycans in all ages of cartilage investigated. The ...
Detection of Salmonella enteritidis in equine feces using the polymerase chain reaction and genus-specific oligonucleotide primers. Salmonella was identified in feces from horses, using the polymerase chain reaction (PCR) and genus-specific oligonucleotide primers. Feces from healthy horses were determined to be culture negative and PCR negative for Salmonella. Fecal samples were inoculated with known numbers of colony-forming units (CFU) of S. enteritidis. The fecal samples were enriched overnight in tetrathionate broth, and then DNA was extracted and amplified by PCR using genus-specific primers. Sensitivity of the assay extended to 10 degrees CFU Salmonella enteritidis/g feces; sensitivity of microbiologic culture with ...
Taxon-specific probes for the cellulolytic genus Fibrobacter reveal abundant and novel equine-associated populations. A total of six 16S rRNA targeted oligonucleotide probes were used to quantify Fibrobacter abundance and diversity in the gastrointestinal contents of a pony. Approximately 12% of the total 16S rRNA extracted from cecal contents hybridized with a Fibrobacter genus-specific probe and a Fibrobacter succinogenes species-specific probe. However, no significant hybridization was observed with a probe for the species. Fibrobacter intestinalis or with three probes for F. succinogenes subspecies. This suggested the presence of a previously undescribed population of F. succinogenes-like organisms. Novel...
Delineating minimal protein domains and promoter elements for transcriptional activation by lentivirus Tat proteins. Lentivirus Tat proteins comprise a novel class of RNA-binding transcriptional activators that are essential for viral replication. In this study, we performed a series of protein fusion experiments to delineate the minimal protein domains and promoter elements required for Tat action. We show that a 15-amino-acid region of equine infectious anemia virus (EIAV) Tat protein, when fused to the GAL4 or LexA DNA binding domain, can activate transcription in appropriate promoter contexts. In the natural human immunodeficiency virus type 1 long terminal repeat, activation by Tat is dependent on multi...
Differences in a ribosomal DNA sequence of Strongylus species allows identification of single eggs. In the current study, molecular techniques were evaluated for the species identification of individual strongyle eggs. Adult worms of Strongylus edentatus, S. equinus and S. vulgaris were collected at necropsy from horses from Australia and the U.S.A. Genomic DNA was isolated and a ribosomal transcribed spacer (ITS-2) amplified and sequenced using polymerase chain reaction (PCR) techniques. The length of the ITS-2 sequence of S. edentatus, S. equinus and S. vulgaris ranged between 217 and 235 nucleotides. Extensive sequence analysis demonstrated a low degree (0-0.9%) of intraspecific variation...
Development of PCR assays to detect genetic variation amongst equine herpesvirus-1 isolates as an aid to epidemiological investigation. A search for variable restriction sites has been carried out for equine herpesvirus-1 (EHV-1) in an attempt to develop markers which can be used to group epidemiologically related viruses into groups, and to learn more about the dynamics of EHV-1 disease. Crude viral DNA extracts of EHV-1, prepared by Hirt extraction, were digested with AluI, HaeIII, or RsaI, and Southern blotted following electrophoresis. DNA fingerprints, produced by probing the Southern blots with the EHV-1 EcoR1-I fragment, separated 56 isolates into 16 groups. The variable sites within the EcoR1-I fragment were mapped app...
Catecholamine affects acetylcholine release in trachea: alpha 2-mediated inhibition and beta 2-mediated augmentation. We investigated the effects of catecholamines on acetylcholine (ACh) release from equine airway parasympathetic nerves. Trachealis strips were suspended in 2-ml tissue baths with Krebs-Henseleit solution containing atropine (10(-7) M), neostigmine (10(-6) M), and guanethidine (10(-5) M). Electrical field stimulation (20 V, 0.5 ms, 0.5 Hz, for 15 min) was applied, and ACh was measured by high-performance liquid chromatography with electrochemical detection. Epinephrine (Epi) and norepinephrine (NE) inhibited ACh release in a concentration-dependent manner. Inhibition was attenuated by the alpha...
Enhanced sensitivity to neutralizing antibodies in a variant of equine infectious anemia virus is linked to amino acid substitutions in the surface unit envelope glycoprotein. Serial passage of the prototype (PR) cell-adapted Wyoming strain of equine infectious anemia virus (EIAV) in fetal donkey dermal (FDD) rather than fetal horse (designated fetal equine kidney [FEK]) cell cultures resulted in the generation of a variant virus strain which produced accelerated cytopathic effects in FDD cells and was 100- to 1,000-fold more sensitive to neutralizing antibodies than its parent. This neutralization-sensitive variant was designated the FDD strain. Although there were differences in glycosylation between the PR and FDD strains, passage of the FDD virus in FEK cells di...
Regulation of granule size in human and horse eosinophils by number of fusion events among unit granules. 1. We have investigated the granule size distributions in human and horse eosinophils by time-resolved patch-clamp capacitance measurements. 2. During exocytosis of single granules the electrical capacitance of the plasma membrane increases in discrete steps. The steps in horse cells are about six times larger than those in human cells in accordance with the difference in granule size. 3. In both species a multimodal capacitance step size distribution is observed with a first peak at 6-7 fF corresponding to granules with a diameter of about 450-500 nm and a surface area of about 0.7 microns2, ...
The proximal ligand variant His93Tyr of horse heart myoglobin. The spectroscopic and structural properties of the His93Tyr variant of horse heart myoglobin have been studied to assess the effects of replacing the proximal His residue of this protein with a tyrosyl residue as occurs in catalases from various sources. The variant in the ferric form exhibits electronic spectra that are independent of pH between pH 7 and 10, and it exhibits changes in absorption maxima and intensity that are consistent with a five-coordinate heme iron center at the active site. The EPR spectrum of the variant is that of a high-spin, rhombic system similar to that reported for...
Characterisation of a membrane receptor on ruminants and equine platelets and peripheral blood leukocytes similar to the human integrin receptor glycoprotein IIb/IIIa (CD41/61). This paper describes two anti-glycoprotein IIb/IIIa or CD41/61 murine monoclonal antibodies (Co.35E4 and Co.2oA1). The cellular distribution and apparent molecular weight of the antigen detected by these antibodies is consistent with their reaction with ruminant and equine glycoprotein IIb/IIIa. Biochemical analysis of the equine molecule using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed bands of 24, 100 and 110 kDa under reducing conditions and 115 and 80 kDa under nonreducing conditions. Biochemical analysis of ruminant antigen revealed that the 24 kDa band...
Electrostatics of hemoglobins from measurements of the electric dichroism and computer simulations. Hemoglobins from normal human cells, from sickle cells, and from horse were investigated by electrooptical methods in their oxy and deoxy forms. The reduced linear dichroism measured as a function of the electric field strength demonstrates the existence of permanent dipole moments in the range of 250-400 Debye units. The reduced limiting dichroism is relatively small (< or = 0.1); it is negative for hemoglobin from sickle cells and positive for the hemoglobins from normal human cells and from horse. The dichroism decay time constants are in the range from about 55 to 90 ns. Calculations of th...
Production of monoclonal antibodies in horses. Monoclonal antibodies (MAbs) have been successfully used to evaluate immune responses in horses, and to target important antigens of equine infectious agents to which protective immune responses may be directed (1–5). Most of these studies are performed with murine MAb produced by fusing spleen cells from immunized mice with an appropriate myeloma cell line, as described in Chapter 3. However, there are experimental questions for which murine MAb are not adequate. These include:
1.Definition of microbial epitopes recognized by the infected host;
2.Identification of immunodominant epito...
Molecular analysis of an XY mare with gonadal dysgenesis. In this study, cytogenetic analysis of an infertile mare revealed a 64, XY karyotype. The XY sex-reversed animal had a female phenotype with gonadal dysgenesis. Using Southern blot analysis, we tested for the presence of two Y-specific genes SRY and ZFY by using DNA isolated from peripheral blood leukocytes. The results showed that at least the DNA-binding domain of the SRY gene was deleted from the Y chromosome of the XY mare but that the ZFY gene was present on this chromosome.
Isolation and identification of two potent neurotoxins, aspartic acid and glutamic acid, from yellow star thistle (Centaurea solstitialis). Horses grazing for prolonged periods on yellow star thistle (YST), a plant which grows wild in western parts of the United States, develop an extrapyramidal disorder known as nigropallidal encephalomalacia (NPE). Attempts have been made to identify, isolate, and characterize the toxins responsible for the disease in animals. Using the organotypic tissue culture system on mouse cortical explants as a specific assay method for neurotoxicological evaluation, it has been possible to isolate and characterize two potent neuroexcitotoxic compounds, aspartic and glutamic acids, the former being the ma...
Genotyping of isolates of Taylorella equigenitalis from thoroughbred brood mares in Japan. Profiles of the genomic DNA of 104 strains of T. equigenitalis isolated from brood mares with contagious equine metritis in Hokkaido during the breeding seasons from 1980 to 1993, as well as those of five strains (SS28, EQ56, EQ59, EQ70 and HH139) previously isolated in Japan were examined after restriction digestion and crossed-field gel electrophoresis. These profiles were essentially identical to each other and the various isolates and strains appeared to have a common genotype, designated 'genotype J', with respect to two restriction enzymes, ApaI and NotI. These results suggest a common s...
Continuous in vitro cultivation of erythrocytic stages of Babesia equi. The protozoan parasite Babesia equi, a causative agent of equine piroplasmosis, was continuously cultivated in horse erythrocytes. The parasites were isolated from a carrier horse at a time when no parasite was detected in a thin blood smear. The culture medium consisted of modified medium 199 supplemented with 40% non-heat-inactivated horse serum in a humidified atmosphere containing 5% CO2, 2% O2, and 93% N2 at 37 degrees C. Parasites were detected after 2 days in culture. When the percentage of parasitized erythrocytes (PPE) reached 1%, the cultures were transferred into a humidified atmosp...
Equine parafilariosis in Iran. From March to September 1991, horses (n = 1567), donkeys (n = 112) and mules (n = 96) were examined for equine parafilariosis by random sampling from different areas of Iran. The clinical signs of equine parafilariosis were observed in 136 cases of two northern areas (1.4% and 41.3% infection rate) and one northeastern area (6.3% infection rate). Most of the infected cases were confirmed by laboratory findings. All infected cases were followed up to establish the epidemiological features. The results suggest that there is one enzootic area in Iran, the Caspian coast and Persian Turkman steppes...
Laboratory diagnosis of equine pituitary pars intermedia adenoma. The objective of the study was to determine the sensitivity with which an adenoma of the pars intermedia of the pituitary gland can be predicted in horses by measuring the basal concentrations of glucose, cortisol, adrenocorticotropin (ACTH), and insulin, the urinary concentration of corticoids, the urinary corticoid:creatinine ratio, and the plasma cortisol concentration after the administration of 25 IU of ACTH intravenously. The records of 24 cases of histologically confirmed equine pituitary pars intermedia adenomas were used. An adenoma of the pars intermedia of the pituitary gland in hor...
Pharmacokinetics of intravenously administration of prednisolone in the horse as determined by radioimmunoassay. A radioimmunoassay was developed for prednisolone using IgG purified from rabbit antiserum. The assay was employed to determine the pharmacokinetics of prednisolone following intravenous administration of 450 mg of prednisolone sodium succinate (Solu Delta Cortef) to five adult Thoroughbred horses. The RIA had a sensitivity of 2 ng/ml and was relatively specific. It had cross-reactivity with 21-deoxycortisol (83.3%) cortisol (27.8%), 11-beta-hydroxyprogesterone (39.2%) and 17-hydroxyprogesterone (50%). However, it did not cross-react with naturally occurring steroids (cholesterol, testosterone...
Characterization of a density-corrected ultrasonic pneumotachometer for horses. A density-corrected ultrasonic pneumotachometer designed specifically for horses (UF202) was evaluated and characterized with the aid of a custom-built apparatus. UF202 provided voltage outputs for airflow through and gas density within the flowhead. Baseline stability for flow channel output (VUF202) was or = 0.9976). Under optimal conditions, VUF202 accuracy was determined to be +/- 1.00% FS and repeatability was +/- 0.78% FS. VUF202 resolution was 24 ml/s. The rise time for VUF202 was 18 ms, and the -3-dB point was 18 Hz; digital compensation provided a flat frequency response to 32 Hz. VU...
D-dimer improves the prognostic value of combined clinical and laboratory data in equine gastrointestinal colic. The discriminating ability of 15 parameters alone or in combinations, including results from analysis of plasma endotoxin, the Nycomed plasma D-Dimer test and phospholipase A2, were analyzed to predict morbidity and mortality in equine gastrointestinal colic. Endotoxaemia was a characteristic feature of the colic horses. The problem of adequately predicting nonsurvivors among colic horses required several parameters to be included in the logistic model: if the "classical parameters", (heart rate, respiratory rate, PCV, anion gap) were included in the model, addition of plasma D-dimer, phosphol...
The hemostatic profile of equine ovarian follicular fluid. The coagulation factors VII and X and fibrinogen were detected in equine ovarian follicular fluid. The amounts of fibrinogen and factor X were approximately 40 percent of that found in normal equine plasma while the level of factor VII was lower, at approximately 14 percent. The addition of human recombinant tissue factor caused fibrin formation in the follicular fluid. The thrombin generating activity appears to be confined to the tissue factor pathway since no activity associated with factors VIII:C, IX or IX was detected. Fibrinolytic activity, at higher levels than that found in plasma, wa...
The nucleotide sequence of asinine herpesvirus 3 glycoprotein G indicates that the donkey virus is closely related to equine herpesvirus 1. The nucleotide sequence of the glycoprotein G (gG) homologue of asinine herpesvirus 3 (AHV3), a respiratory alphaherpesvirus of donkeys, was determined. The AHV3 gG gene consists of 1233 base pairs (bp) and codes for a predicted protein of 411 amino acids. This is identical in size to the equine herpesvirus 1 (EHV1) gG gene and 6 amino acids longer than the equine herpesvirus 4 (EHV4) gG gene. The predicted amino acid sequence of AHV3 gG has characteristics of a class 1 membrane protein. The amino acid sequence of AHV3 gG shows 92% and 60% identity to EHV1 gG and EHV4 gG respectively. Two regi...
Detection of equine arteritis virus (EAV) by polymerase chain reaction (PCR) and differentiation of EAV strains by restriction enzyme analysis of PCR products. A polymerase chain reaction (PCR) based assay capable of detecting and differentiating seven strains of equine arteritis virus (EAV) from around the world was developed. The primers for the PCR were chosen from the ORF6 gene encoding the unglycosylated membrane protein (M). Viral RNA from cell culture fluids infected with each of the seven EAV strains and RNA from the live vaccine, Arvac, was detected by PCR using four sets of primers. The sensitivity of detection was increased from 100 to 1,000 times by performing nested PCR enabling the detection of RNA at a level of 0.5-5 PFU. Differentiati...
Enzyme-linked immunosorbent assay for myosin heavy chains in the horse. The content in slow and fast myosin heavy chains (MHC 1 and MHC 2) of 5 equine muscles was determined using an enzyme-linked immunosorbent assay. The results obtained with this immunoenzymatic method were compared with complementary techniques: electrophoresis and immunohistochemistry. Slices of masseter, diaphragm, tensor faciae latae, semitendinosus and cutaneus trunci were obtained from a 12-year-old saddle horse after slaughter. Muscular proteins were specifically extracted to be analysed by ELISA. The technique used 2 complimentary monoclonal antibodies (MAb). MAb 1 was prepared from a hu...
