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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.
Characteristics of equine herpesvirus 1 glycoproteins expressed in insect cells. A series of recombinant baculoviruses containing genes for glycoproteins C, D, H and L of equine herpesvirus 1 (EHV-1) have been constructed, and the EHV-1 products characterised by gel electrophoresis and immunoblotting. The EHV-1 glycoproteins expressed in insect cells were similar but not identical in apparent sizes to those expressed in EHV-1 infected mammalian cells. Each of the EHV-1 products was recognised by convalescent equine sera, indicating that they were all targets for an equine immune response. Mice immunised with baculovirus-expressed EHV-1 gD and gC acquired an enhanced abilit...
Amino acid sequence of HSP-1, a major protein of stallion seminal plasma: effect of glycosylation on its heparin- and gelatin-binding capabilities. We report the complete amino acid sequence of HSP-1, a major protein isolated from stallion seminal plasma or acid extracts of ejaculated spermatozoa. The protein consists of 121 amino acids organized in two types of homologous repeats arranged in the pattern AA'BB'. Each of the 13-15-residue A-type repeats contains two O-linked oligosaccharide chains. The B-type repeats span 44-47 amino acids each, are not glycosylated, and have the consensus pattern of the gelatin-binding fibronectin type-II module. This domain also occurs in the major bovine seminal plasma heparin-binding proteins PDC-109 (...
Activities of selected aerobic and anaerobic enzymes in the gluteus medius muscle of endurance horses with different performance records. Biopsies of the gluteus medius muscle were taken at three different depths from 36 endurance horses aged 8.42 +/- 2.85 years and of both sexes. Twenty of the horses were considered to be excellent performers on the basis of the mean speed of their three fastest records in endurance events over the previous two or three years, whereas 16 were moderate performers. The biopsy samples were analysed for the activities of the enzymes citrate synthase (an indicator of citric acid cycle activity), 3-OH-acyl-CoA-dehydrogenase (an indicator of lipid oxidation) and lactate dehydrogenase (an indicator of ...
Partial sequence of the equine immunoglobulin epsilon heavy chain cDNA. In order to isolate a part of the immunoglobulin E (IgE) heavy chain cDNA of the horse, primers have been designed based upon well conserved sequences in humans, sheep and rats. The PCR resulted in a 500 bp fragment which hybridised with a human IgE constant region probe. The fragment was cloned and sequenced and its derived protein sequence compared with the corresponding sequences in humans, sheep and mice. Most amino acids common to these three species are also shared by the horse.
Detection of African horse sickness viruses by dot-blot hybridization using a digoxigenin-labelled probe. In order to develop a non-radioactive dot-blot hybridization assay, for the detection of African-horse sickness virus (AHSV), genome segment 7 from 9 serotypes was amplified by RT-PCR. The resulting PCR products were denatured, immobilized on nylon membranes and then hybridized to a non-radioactive digoxigenin-labelled probe. This probe (265 bp in length) was generated by nested-PCR using genome segment 7 of AHSV, serotype 4 as a template. The dot-blot was visualized by chemiluminescence. Positives were obtained from the PCR products amplified from all 9 AHSV serotypes, but not from any other ...
Comparison of time-resolved fluoroimmunoassay and immunoenzymometric assay for clenbuterol. A time-resolved fluoroimmunoassay (TR-FIA) for the direct determination of clenbuterol residues in horse urine using a highly specific monoclonal antibody has been compared with an immunoenzymometric assay (IEMA). The sensitivity of both methods was 10 pg; the calibration curve was linear between 10 and 10(5) pg for the TR-FIA and between 10 and 10(4) pg for the IEMA.
Localisation of alkaline phosphatase in equine growth cartilage. The aim of this study was to localise alkaline phosphatase (ALP) activity in equine growth cartilage both histochemically and ultrastructurally. For histochemical studies, full thickness growth cartilage samples were obtained from 6 anatomical sites from 16 horses and ponies ranging in age from 90 d postconception to 12 years of age. For ultrastructural studies, samples were obtained from the lateral trochlear ridge of the distal femur of 3 animals ranging in age from 157 d postconception to 12 months of age. Alkaline phosphatase in histological sections was localised using a substituted napht...
Detection of immunoreactive atrial and brain natriuretic peptides in the equine atrium. The distribution of immunoreactivity (IR) for cardiodilatin/atrial natriuretic peptide (CDD/ANP) and brain natriuretic peptide (BNP) was examined immunohistochemically and immuno-electron-microscopically in the equine atrium, using specific antibodies. In the immunohistochemical studies, IR-CDD/ANP and IR-pBNP-26 (porcine BNP-26 immunoreactivity) was detected in the cytoplasm of the auricular cardiocytes, but IR-hBNP-32 (human BNP-32 immunoreactivity) was not. The double immunogold labelling method for IR-hBNP-28 and IR-pBNP-26 revealed that gold particles of different sizes were located in th...
Subcellular biochemical changes during the development of the small intestine of pony foals. To examine the postnatal development of equine small intestine, biopsy specimens of jejunal mucosa from 8 ponies, between 6 and 28 weeks old, were subjected to analytical subcellular fractionation and assay of organelle marker enzymes. Fractionation revealed a reduction in the particulate brush border component of beta-galactosidase (lactase) activity between 6 and 28 weeks, and a corresponding increase in soluble activity, although the reduction in mean specific activity was not significant. There also was a decrease in the proportion of brush border to soluble aminopeptidase N activity, a re...
Equine arteritis virus-neutralizing antibody in the horse is induced by a determinant on the large envelope glycoprotein GL. Complementary DNAs encoding ORFs 2 to 7 equine arteritis virus (EAV) have been cloned into the expression vector pGEX to produce glutathione-S-transferase fusion proteins. Recombinant proteins were affinity purified and screened in ELISA with equine sera to identify immunoreactive polypeptides. The large envelope glycoprotein (GL) was identified as the most reactive to EAV-positive equine sera and an immuno-dominant epitope was mapped between amino acids 55 and 98 by subcloning and expression. A fusion protein covering this region and a GL-specific synthetic peptide (residues 75 through 97) in...
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...
