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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.
Pharmacokinetics of heparin and its pharmacodynamic effect on plasma lipoprotein lipase activity and coagulation in healthy horses. We evaluated the pharmacokinetics of IV administered sodium heparin and the pharmacodynamic effect of heparin on lipoprotein lipase (LPL) activity. Horses were allotted to 3 groups. Plasma samples were obtained from each horse before and at various times for 6 hours after heparin administration for determination of heparin concentration, LPL activity, and activated partial thromboplastin time (APTT). The disposition of heparin was dose dependent. The area under the plasma heparin concentration vs time curve (AUC) increased more than proportionally with dose, indicating that heparin elimination...
Monoclonal antibodies specific for equine IgG sub-isotypes including an antibody which recognizes B lymphocytes. Equine immunoglobulin G is currently classified as consisting of five sub-isotypes: IgGa, b, and c, IgG(T), and IgG(B). The study of the role of these immunoglobulins in antigen-specific responses, and the examination of their functional properties would be greatly facilitated by the availability of monoclonal antibodies (Mabs) that distinguish between them. The production and characterization of two Mabs that recognize an IgG sub-isotype with the characteristics of IgG(ab) is described. The immunoglobulin identified by these Mabs had a heavy chain weight of 53 kDa, was of rapid cathodal elect...
Epitope mapping of cross-reactive monoclonal antibodies specific for the influenza A virus PA and PB2 polypeptides. Characterization of the epitopes recognized by 21 monoclonal antibodies (MAbs) specific for the influenza A virus PA (13 MAbs) and PB2 (8 MAbs) polypeptides (Bárcena et al. (1994) J. Virol. 68, 6900-6909) raised against denatured polypeptides produced in E. coli is described. MAbs were characterized by: (1) competitive binding ELISAs; (2) mapping of the protein regions that specify their binding sites; and (3) analyses of their ability to recognize the corresponding viral protein in a number of viral isolates. Five and three non-overlapping antigenic areas were defined by the anti-PA and anti...
Incorporation of uracil into viral DNA correlates with reduced replication of EIAV in macrophages. The retrovirus equine infectious anemia virus (EIAV) encodes a dUTPase situated between reverse transcriptase and integrase. We have described the inability of EIAV with a 270-bp dUTPase deletion, delta DU EIAV, to replicate to wild-type (WT) levels in equine macrophages (D. S. Threadgill, W. K. Steagall, M. T. Flaherty, F. J. Fuller, S. T. Perry, K. E. Rushlow, S. F. J. LeGrice, and S. L. Payne, J. Virol. 67, 2592-2600, 1993). Here we describe the construction of a second dUTPase-deficient virus (DUD71E) containing a single amino acid substitution in dUTPase. delta DU and DUD71E replicate to ...
Needle aspiration lung biopsy: reevaluation of the blood patch technique in an equine model. To reexamine the blood patch technique in a laboratory model of lung biopsy free of confounding clinical variables. Methods: An equine model of lung biopsy was developed with an excised lobe connected to an insufflation bulb and pressure monitor. Patched and control unpatched punctures were made in the lung surface, and the pressure within the lung was raised to the maximum achievable. Whether air leakage from the puncture sites could be induced was determined and, if so, at what pressure it occurred. Results: At statistical analysis with the Kaplan-Meier test and the Cox proportional hazards ...
Monoclonal equine IgM and IgG immunoglobulins. In order to define equine immunoglobulins (Igs) and to produce monoclonal reference Igs we fused equine peripheral blood mononuclear cells with X63-Ag8.653 non Ig producing murine myeloma cells. A total of 29 equine Ig producing equi-murine heterohybridomas were obtained, of which ten expressed equine Ig for more than 3 months. One of these heterohybridoma lines produced monoclonal IgM, an equine isotype which has not been available in monoclonal form before. Four lines secreted equine IgG of two distinct Ig heavy chain types as assessed by the molecular weight (MW), while the remaining five l...
Volume changes of the molten globule transitions of horse heart ferricytochrome c: a thermodynamic cycle. Volume changes among the unfolded (U), native (N), and molten globule (MG) conformations of horse heart ferricytochrome c have been measured. U to N (pH 2 to pH 7) was determined in the absence of added salt to be -136 +/- 5 mL/mol protein. U to MG (pH 2, no added salt to pH 2, 0.5 M KCl) yielded + 100 +/- 6 mL/mol. MG to N was broken into two steps, N to NClx at pH 7 by addition of buffered KCl to buffered protein lacking added salt (NClx = N interacting with an unknown number, X, of chloride ions), and MG to NClx by jumping MG at pH 2 in 0.5 M KCl to pH7 at the same salt concentration. The d...
Development and evaluation of an ELISA using recombinant fusion protein to detect the presence of host antibody to equine arteritis virus. A recombinant glutathione-S-transferase fusion protein expressing amino acids 55-98 of equine arteritis virus (EAV) GL (rGL 55-98) was tested in an ELISA for its ability to detect serum antibodies to EAV. Host antibodies induced following EAV infection bound the recombinant antigen by ELISA. The ELISA specificity and sensitivity were determined with a panel of equine sera including postinfection and postvaccination samples. A good correlation existed between EAV neutralizing antibody titers and ELISA absorbance values (r = 0.827). The sensitivity and specificity of the ELISA were 99.6 and 90.1...
Assessment of glycosaminoglycan concentration in equine synovial fluid as a marker of joint disease. A modification of a colorimetric assay was used to determine synovial fluid total and individual sulphated-glycosaminoglycan concentration in various clinical presentations of joint disease in horses. Concentrations of synovial fluid and serum sulphated-glycosaminoglycan (GAG) were measured by the 1,9-dimethylmethylene blue (DMMB) dye assay in normal horses (n = 49), horses with acute (n = 26) or chronic (n = 27) joint disease (defined by clinical, radiographic, and clinicopathological parameters), and horses with cartilaginous lesions at diagnostic arthroscopy, but with normal radiographs and...
In vitro and in vivo evidence on the site of neutralization of equine chorionic gonadotrophin (eCG) by an eCG antiserum. This study was designed to determine whether the major site of eCG neutralization by an antiserum to the hormone is at the peripheral or ovarian level. Hamsters hypophysectomized at oestrus were injected s.c. with 25 iu eCG. Three days later, preovulatory follicles were dissected and cultured for 5 h and the medium was changed every hour. At the end of the first hour of incubation, oestradiol and androstenedione accumulation was high, with a sharp drop over the next 4 h, whereas progesterone concentrations did not change over the entire period. Addition of eCG antiserum to the incubated follic...
A mouse model for testing the pathogenicity of equine herpes virus-1 strains. A mouse model was developed for testing the pathogenicity of equine herpes virus-1 (EHV-1) strains. The model was validated with EHV-1 strains that are known to be of a low or high pathogenicity in horses. From all parameters tested, the safety index, which was calculated from the body weights of the mice after infection, proved to be the best predictive parameter. When this parameter was used, good and reliable correlations were found with the pathogenicity of the EHV-1 strains in horses. This method enabled the differentiation between the two experimental EHV-1 strains whose genetic backgrou...
A lectin histochemical study of gustatory (von Ebner’s) glands of the horse tongue. In the present work, gustatory glands (von Ebner's glands) of the horse tongue were examined by means of five peroxidase-conjugated lectins (PNA, DBA, SBA, UEA I, WGA), with and without prior sialidase digestion, in order to investigate the presence and distribution of carbohydrate residues in secretory cells and duct cells. The most intense staining of secretory cells was observed with PNA after pre-treatment with neuraminidase. This indicates that the terminal trisaccharide sequence sialic acid- (alpha 2-->3, 6) galactosyl (beta 1-->3) N-acetylgalactosamine is the most frequent oligosa...
At least four MHC class I genes are transcribed in the horse: phylogenetic analysis suggests an unusual evolutionary history for the MHC in this species. Nineteen horse MHC class I specificities have been serologically identified previously at a single locus (ELA-A), and two other specificities appear to be coded at other loci. Biochemical studies indicate that there are at least two expressed loci. In order to establish the number of transcribed horse MHC class I genes, we made a cDNA library from a heterozygous animal (ELA-A3/A7), and screened for positive clones using a bovine class I probe. More than 200 class I clones were isolated in this way, and so far seven unique full length sequences have been identified. All of the sequences are pre...
The relationship between single radial hemolysis, hemagglutination inhibition, and virus neutralization assays used to detect antibodies specific for equine influenza viruses. Antibodies specific for equine influenza viruses are usually quantified using single radial hemolysis (SRH), hemagglutination inhibition (HI) or virus neutralization (VN). Neutralizing antibodies are thought to provide optimum protection to challenged animals. The purpose of this study was to determine the extent to which SRH and HI assays detect antibodies which neutralize equine influenza viruses. Acute and convalescent sera from 41 horses were analyzed using VN, SRH, and HI assays. These horses were present in a population of Thoroughbred racehorses during an epidemic of upper respiratory t...
Serum protein concentrations in horses with severe liver disease: a retrospective study and review of the literature. The present retrospective study was undertaken to determine the frequency of hypoproteinemia and hypoalbuminemia in horses with natural occurring severe liver disease. The study represents a review of case records and laboratory data of 84 horses presented with acute or chronic liver disease to the University of California Veterinary Medical Teaching Hospital between 1973 and 1991. Forty horses (48%) had serum protein concentrations above the maximum reference value (7.7 g/dL). The increase in serum protein concentration was associated with hyperglobulinemia (P = .00005, R2 = .80). Only 13% (1...
Comparative RAPD-PCR analysis of lungworms (Dictyocaulidae) from fallow deer, cattle, sheep, and horses. Genomic DNA isolated from the four Dictyocaulus species D. viviparus, D. eckerti, D. filaria and D. arnfieldi was compared by random amplified polymorphic DNA polymerase chain reaction (RAPD)-PCR to get additional information whether lungworms from fallow deer belong to a separate species (D. eckerti) or have to be regarded as an isolate of D. viviparus in wild ruminants. The resulting banding patterns of the electrophoresed PCR products were compared to assess the degree of genetic differences between the different lungworms. For the two D. viviparus isolates a similarity coefficient of 93.4%...
Determination of triamcinolone acetonide in equine serum and urine by liquid chromatography-atmospheric pressure ionization mass spectrometry. Urine and serum samples collected from four standard-bred mares after 30-mg intraarticular administrations of triamcinolone acetonide were analyzed using combined high-performance liquid chromatography-atmospheric pressure ionization mass spectrometry. Maximum triamcinolone acetonide concentrations of 32.3, 14.8, 24.3, and 29.4 ng/mL in the urine and 2.7, 1.9, 2.3, and 2.5 ng/mL in the serum samples were observed. The peak concentrations of the drug were detected approximately 22 h (urine) and 12 h (serum) after administration. The drug elimination profiles for both urine and serum are present...
Detection of clenbuterol (Ventipulmin) in the horse. An enzyme linked immunosorbent assay (ELISA) was developed to detect the beta 2-agonist clenbuterol in equine blood and urine. The antiserum was raised in rabbits, employing clenbuterol-diazo-BSA as antigen. Clenbuterol-diazo-horseradish peroxidase served as enzyme conjugate. The concentration of clenbuterol to decrease tracer binding by 50% (IC50 value) was found to be 27.50 +/- 4.20 pg/well (1.37 ng/ml). The antibody cross-reacted with salbutamol (30%), terbutaline (14%) and cimaterol (1%). Horse serum was used directly to screen for clenbuterol, while urine was employed diluted. Positive sc...
Tropane alkaloids and toxicity of Convolvulus arvensis. Horses in a few, localized northern Colorado pastures exhibited weight loss and colic. At post mortem, intestinal fibrosis and vascular sclerosis of the small intestine was identified. The pastures where the affected horses grazed were overrun by field bindweed (Convolvulus arvensis). Bindweed from the pasture was found to contain the tropane alkaloids tropine, pseudotropine, and tropinone and the pyrrolidine alkaloids cuscohygrine and hygrine. Laboratory mice readily ate C. arvensis and exhibited a variety of abnormal clinical signs depending on the amount eaten. Similar alkaloids have been f...
Application of the polymerase chain reaction to the detection of African horse sickness viruses. The development of a coupled reverse transcriptase-polymerase chain reaction assay (RT-PCR) is described for the detection of African horse sickness virus (AHSV) double-stranded RNA. Genome segments 7 and 10 were chosen as target templates for primers selected for use in the RT-PCR. Using these AHSV-specific primers all 9 serotypes were detectable. The sensitivity and specificity of the RT-PCR results were compared to those obtained by competition ELISA.
Regulatory function of the equine herpesvirus 1 ICP27 gene product. The UL3 protein of equine herpesvirus 1 (EHV-1) KyA strain is a homolog of the ICP27 alpha regulatory protein of herpes simplex virus type 1 (HSV-1) and the ORF 4 protein of varicella-zoster virus. To characterize the regulatory function of the UL3 gene product, a UL3 gene expression vector (pSVUL3) and a vector expressing a truncated version of the UL3 gene (pSVUL3P) were generated. These effector plasmids, in combination with an EHV-1 immediate-early (IE) gene expression vector (pSVIE) and chimeric EHV-1 promoter-chloramphenicol acetyltransferase (CAT) reporter constructs, were used in trans...
Development of an ELISA using a universal method of enzyme-labelling drug-specific antibodies. Part I: Detection of dexamethasone in equine urine. The development, validation, and application of an ELISA for dexamethasone in equine urine is described. The drug-protein conjugate was immobilised in microtitre plate wells and antiserum raised against the same drug-protein conjugate was allowed to compete with sample or standard drug and the immobilised drug-protein conjugate. The proportion of antiserum binding to the immobilised drug-protein conjugate was detected using a biotinylated protein G/extravidin-alkaline phosphatase complex in situ and measurement of the substrate product. The method was used to detect the presence of drug-derive...
A novel Ca(2+)-dependent step in exocytosis subsequent to vesicle fusion. Exocytosis begins with formation of a small fusion pore which then expands allowing rapid release of granular contents. We studied the influence of cytoplasmic free Ca2+ ([Ca2+]i) on the conductance of the initial pore and on the dynamics of subsequent expansion in horse eosinophils using the patch clamp technique. The mean initial conductance is approximately 200 pS independent of [Ca2+]i. This value is close to that previously found in beige mouse mast cells. The pore subsequently expands by 18 nS/s at [Ca2+]i < 10 nM, by 40 nS/s at [Ca2+]i = 1.5 microM and by 90 nS/s at [Ca2+]i = 10 micr...
Substitutions of isoleucine residues at the adenine binding site activate horse liver alcohol dehydrogenase. The contributions of isoleucine residues 224 and 269 of horse liver alcohol dehydrogenase to binding of the adenine moiety of NAD and to catalysis were studied by replacing Ile-224 with glycine (I224G) and Ile-269 with serine (I269S). The kinetic mechanisms of wild-type and both mutated liver enzymes were ordered. Affinities for several adenosine derivatives were decreased 5-50-fold by both substitutions. The I269S mutation differentially destabilized binding of the complete coenzyme, as affinities for NAD+ and NADH were decreased about 60-fold with the I224G enzyme and 350-fold for the I269S ...
The disposition of gentamicin in equine plasma, synovial fluid and lymph. Plasma (P), synovial fluid (SF) and lymph (L) concentrations of gentamicin were studied in two trials. A lymph vessel in the hindlimb was cannulated. The day after surgery (trial A), P and L samples were collected for 12 h after intravenous injection of gentamicin sulphate at 2.2 mg/kg dose rate. Approximately 48 h after surgery (trial B), the fetlock joint of the cannulated hindlimb was catheterized and P, SF and L samples collected for 12 h after a similar intravenous injection. The kinetic parameters were similar to those in other reports and did not differ between trials (P < 0.05). The P,...
Responsiveness of equine basilar artery to transmural nerve stimulation differs from that of porcine and bovine basilar arteries in vitro. Transmural nerve stimulation (TNS) induced relaxations in porcine and bovine basilar arteries which were abolished by tetrodotoxin (TTX) and by L-nitro-arginine (LNAG). However, TNS induced contractions in equine basilar artery which were abolished by TTX and by guanethidine, but not by LNAG. These results suggest that the TNS-induced contractions of equine basilar arteries may be mediated by norepinephrine release.
