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.
Nakajima H, Norcross NL, Coggins L.Antigenic relationship between purified equine infectious anemia (EIA) virus and spleen-derived antigen from EIA-infected horses was examined by immunodiffusion. Identical antigenicity of these two antigens has been proven because precipitation lines formed between the two antigens and EIA antiserum connected with each other. The results indicate that the antigenic substance derived from infected spleen is a component of EIA virus.
Hierholzer JC, Gamble WC, Quist KD, Chappell WA.Horses were immunized by a variety of inoculation procedures designed to determine the most efficient method of producing antisera to adenovirus types 25 to 31. The procedures evaluated included immunization by (i) direct intravenous (iv) injection, (ii) iv infusion, (iii) intramuscular (im) injection of virus with and without Freund's incomplete adjuvant, (iv) combined iv and im injections, and (v) combined iv infusion and im injection. The im schedule (no. 3) was superior to the others in terms of immunizing antigen and time required, and hemagglutination-inhibition (HI) and serum-neutralizi...
Chiewsilp D, McCown JM.Repeated clot formation in mouse ascitic fluids containing antiviral antibody was eliminated by acid precipitation of the fibrinogen.
Gospodarowicz D.A highly purified preparation of PMSG has been obtained from fresh serum and from a commercial preparation. Carbohydrate and amino acid compositions have been determined. The carbohydrate content of PMSG is 46.7% and the molecule is rich in Sialic Acid (13.5%). The apparent molecular weight of PMSG has been determined by SDS polyacrylamide gel electrophoresis. A molecular weight of 53,000 has been found for the unreduced and unalkylated molecule. After reduction and alkylation, the molecular weight fell to 23,000. From these values it has been concluded that PMSG is an oligomeric molecule comp...
Klingeborn B, Dinter Z.The infectivity of equine abortion (herpes) virus (EAV) was inactivated by treatment with reduced dithiothreitol (DTT). According to their susceptibility to DTT, the EAV strains could be divided into three groups. The vaccine strain RAC-H (419) proved to be more resistant to DTT than all of the other 14 strains tested. The hemagglutinin of EAV was also inactivated by DTT; no strain differences were observed in this respect.
Ushimi C, Henson JB, Gorham JR.Primary horse leukocyte cultures were inoculated with 2 or 10 50% tissue culture infective doses (TCID(50)) of equine infectious anemia (EIA) virus per cell, and the titer of cell-associated and fluid-phase virus was determined from 1 to 72 hr postinoculation (PI). Cover slips were collected from 4 to 72 hr PI and stained for EIA viral antigen by the indirect immunofluorescent (FA) technique. Viral replication was detected after a latent period of approximately 18 to 24 hr and reached peak titers of approximately 10(4.5) to 10(6) TCID(50)/0.5 ml from 48 to 72 hr PI. The fluid phase contained 1...
Brunel C, Cathala G.1. Alkaline phosphatases (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) from brain, kidney, liver, bone, lung and spleen, which are not very sensitive to l-phenylalanine, are strongly inhibited by imidazole, whereas the placental and intestinal enzymes, which are very sensitive to l-phenylalanine, are only slightly affected. This is a new possibility for distinguishing the alkaline phosphatase isoenzymes.
2. The inhibition is apparently of an uncompetitive type, suggesting that the inhibitor interacts with the ES complex to form an EIS complex.
3. Histidine acts upon all enzyme...
Barron AL, Caste PG, Paul B, Page LA.Postinoculation sera collected from pigeons, turkeys, guinea pigs, sheep, a calf, a rabbit, and a horse experimentally infected with various strains of Chlamydia psittaci yielded a high incidence of positive reactions when tested by double diffusion in gel. Antigen was a deoxycholate extract of SA-2 strain of C. trachomatis. Good correlation was obtained with results of complement fixation tests, whereas double diffusion in gel was less sensitive. Immunoelectrophoresis of the antigen revealed presence of two antigens in the extract.
Boulanger P, Bannister GL, Carrier SP.An agar-gel immunodiffusion test recommended for the diagnosis of equine infectious anemia was evaluated. Our preliminary observations confirmed those of Coggins concerning the mechanism of the test and the results obtained. Furthermore, emphasis was put on the difficulties encountered in the production of spleen antigens with an optimum amount of reactivity. Acetone-ether extraction procedures for the preparation of a liquid antigen extract are described. This type of antigen was reactive in the complement-fixation test in 1:8 or greater dilution and it is proposed to use the complement-fixat...
Diesing L, Steuber S, Ahmed JS, Hörchner F.The sequential appearance of variable antigen types (VATs) of a clone of Trypanosoma evansi was studied in four ponies. Using luminol-dependent chemiluminescence, VAT populations which had been isolated from parasitemic peaks of single ponies, were tested for specificity with serum samples collected from other ponies. When antibody activity was demonstrated in a combination of trypanosomes and serum, it was concluded that a major VAT appeared in common. In the serum of all animals antibody activity was demonstrated to all VAT populations isolated from the other ponies during the first 4 weeks ...
Houghton E, Copsey J, Dumasia MC, Haywood PE, Moss MS, Teale P.As part of a continuing research program associated with the detection of anabolic steroid residues in horse urine, normal samples from entire male horses have now been investigated. Isomers of three C-18 neutral steroids; 4-estren-17-ol-3-one (1), estrane-3,17-diol (2) and an unsaturated estranediol having a possible structure (3), have been identified in urine samples from two male horses aged 8 and 14 years. Of these three steroids, compound (2) was not detected in the urine of a 2.5 yr old entire male nor in the majority of post-race urine samples from entire male horses average age 3.8 yr...
Yamanouchi K, Yoshida S, Hasegawa T, Ikeda A, Chang KT, Matsuyama S, Nishihara M, Miyazawa K, Takahashi M.cDNA encoding equine inhibin alpha-subunit precursor protein was isolated from an equine ovarian cDNA library. For screening, the DNA probe was amplified by the RT-PCR using primers designed based on the rat inhibin alpha-subunit cDNA sequence. Out of 1.2 x 10(5) plaques screened, 19 positive clones were isolated, and one of these clones (Eq-alpha-11) contained a complete open reading frame encoding 367 amino acids. The similarity of the deduced amino acid sequences of both equine inhibin alpha-subunit precursor protein and the mature protein were greater than 80% to those of other six mammali...
Mykkänen AK, Hyyppä S, Pösö AR, Ronéus N, Essén-Gustavsson B.Monocarboxylate transporter 1 (MCT1) and its ancillary protein CD147 facilitate efflux of lactate from the muscle. Expression of MCT1 and CD147 were studied with immunohistochemistry in type I, IIA, IIAB and IIB fibres of equine gluteal muscle. Staining intensity of MCT1 in the cytoplasm as well as in the membranes of fibre types decreased in the order I=IIA>IIAB>IIB and correlated with the oxidative capacity. Capillaries were pronounced in the MCT1 staining. CD147 antibody stained plasma membranes of all fibre types evenly, whereas the staining in the cytoplasm followed that of MCT1. In...
Sales FA, Ferreira-Silva JC, Vieira JI, Chaves MS, Caldas EL, Filho JP, Freitas VJ, Oliveira MA.Addition of extenders to thawed semen could improve fertility. Objective: To determine the efficiency of extenders to increase viability of thawed semen, measured by sperm parameters in vitro and pregnancy rates after artificial insemination (AI). Methods: Sperm motility and acrosin activity were measured during a thermoresistance test (TRT). Results: Progressive motility decreased (P<0.05) after 30 min in thawing semen treated with saline solution (SS) and only after 60 min with Tyrode's solution (TS) or freezing diluent (FD). The total motility decreased (P<0.05) after 60 min in thawed semen...
Starick E.A reverse transcription-polymerase chain reaction (RT-PCR) assay using four different primer pairs for the detection of equine arteritis virus (EAV) RNA in semen and tissue samples was evaluated. A fragment encoding the leader sequence of the EAV genome was most successfully amplified. The specificity and sensitivity of RT-PCR was assessed by virus isolation in cell culture, restriction analysis, dot blot hybridisation and nested PCR. To this end, 23 semen samples from seropositive stallions and 11 tissue samples from 4 aborted foals were tested. Compared to the virus isolation test in cell cu...
Sayegh AI, Anderson NV, Harding JW, Cerpovicz P, DeBowes RM, Ritter RC, Baker GJ, Reeck G.To purify and characterize pepsinogens in equine gastric mucosa. Methods: Stomachs collected from 2 healthy horses at necropsy. Methods: After collection, stomachs were placed immediately in ice before storage at -48 C. After slow thawing, the mucosa was scraped off while the tissue was immersed in 0.1M potassium phosphate (pH 7.4) at 4 C, then was homogenized. The filtered extract was subjected to anion-exchange chromatography. Fractions that were found to contain pepsin or pepsinogen were further chromatographed. Individual fractions were tested for pepsinogen or pepsin content by monitoring...
Marland A, Sarkar P, Leavitt R.Tenoxicam (Mobiflex) was administered orally to four standardbred mares at a dose of 200 mg. Elimination profiles of tenoxicam and hydroxytenoxicam were generated based on quantitation of these analytes in urine and serum by liquid chromatography (LC) with ultraviolet detection. Tenoxicam was confirmed by LC-tandem mass spectrometry daughter ion mass spectra in the last postadministration sample in which tenoxicam was detected. The tenoxicam and hydroxytenoxicam urinary elimination profiles had the same shape for the same horse; however, each horse was significantly different from the others. ...
Mizobe M, Kondo F, Kumamoto K, Terada T, Nasu H.Rapid and quantitative analytical methods for bilirubin using high-performance liquid chromatography (HPLC) with UV detection were developed for samples from equines at a meat inspection site. Sharp HPLC peaks for bilirubins, unconjugated bilirubin (UCBL) and conjugated bilirubin (CBL), were obtained using a simple mobile phase of methanol:0.5 M Tris-HCl buffer (65:35, v/v, pH 7.4). A variable wavelength detector set at 450 nm, 0.01 AUFS and a recorder set at 4 cm/min were used for detection. Peaks for UCBL and CBL occurred at 7.1 min and 4.9 min, the lower limits of detection ranged between 0...
Mobarak MS, Ryan MF.Adult Strongylus vulgaris, collected from the caecum of infected horses and embedded in paraplast using standard methods, were sectioned for immunohistochemistry (IHC) studies. Antibodies were raised in rabbit against the excretory-secretory product (ESP) and against two constituent protein bands (28-30 kDa). The use of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA) and immunoblotting indicated the immunogenicity of ESP and of the subunits (28-30 kDa). In ELISA, both rabbit hyperimmune sera recognized the ESP and (28-30 kDa) ban...
Brotherton HO, Yost RA.A screening and confirmation procedure for drugs and metabolites in the blood serum and urine of racing animals was developed. Equine blood serum was spiked with low concentrations of several drugs of interest. Canine blood serum and urine were collected following oral doses of diethylcarbamazine, procaine, and phenylbutazone. Serum, urine, and extracts of each were analyzed, using a triple quadrupole mass spectrometer. Simultaneous screening of up to 50 drugs was possible in a single sample, in less than 2 minutes. Detection limits for most compounds were in the ng/ml to microgram/ml range, u...
Bannai H, Nemoto M, Tsujimura K, Yamanaka T, Kokado H, Kondo T.In order to establish an efficient system for serological diagnosis of equine viral arteritis in Japan, we compared enzyme-linked immunosorbent assays (ELISAs) provided by two manufacturers (Nisseiken Co., Ltd., Tokyo, Japan, and VMRD Inc., Pullman, WA, U.S.A.) by testing a series of horse sera. The results revealed that 159 of 160 virus-neutralizing (VN) antibody-positive serum samples were positive in both the Nisseiken-ELISA and VMRD-ELISA. Of the VN-negative sera (n=157), 134 and 154 samples were negative in the Nisseiken-ELISA and VMRD-ELISA, respectively. Sensitivity was 99.4% for both t...
Reubel GH, Studdert MJ.We report the first nucleotide sequence data on equine adenovirus 2 (EAdV2) which corroborate on the molecular level that EAdV2 is distinct from equine adenovirus 1 (EAdV1). Based on sequence homology with Eadv1 the hexon gene of Eadv2 was identified. HindIII restriction fragments containing the hexon and eight other viral genes were cloned into the plasmid pUC19 and the nucleotide sequence of the hexon and the 23K proteinase genes completely determined. Amino acid (aa) comparison of sequence fragments with published adenovirus (AdV) proteins identified the genes for the E1B/19K, IVa2, DNA pol...
Guèrand M, Mahla R, Lagneaux D, Amigues Y, Palmer E, Bézard J.Paternity analysis was performed on the DNA of 21 equine embryos collected nonsurgically 10 days after ovulation from known mares, but involving 3 possible sires. After extraction, the DNA of each embryo was typed by radioactive PCR amplification using 10 characterised microsatellites; HMS 1, 2, 5, 6, 7 and 8 (Guérin et al. 1994) and HTG 3, 4, 6 and 10 (Marklund et al. 1994). The 21 dams and 3 sires were genotyped using DNA extracted from blood and amplified by PCR. After electrophoresis and autoradiography of the PCR products of the embryo and parents, the alleles of the embryo were compared...
Vonaparti A, Lyris E, Panderi I, Koupparis M, Georgakopoulos C.Two simple and rapid LC/MS methods with direct injection analysis were developed and validated for the quantification and identification of hydrocortisone in equine urine using the same sample preparation but different mass spectrometric systems: ion trap mass spectrometry (IT-MS) and time-of-flight mass spectrometry (TOF-MS). The main advantage of the proposed methodology is the minimal sample preparation procedure, as particle-free diluted urine samples were directly injected into both LC/MS systems. Desonide was used as internal standard (IS). The linear range was 0.25-2.5 microg ml(-1) for...
Richa , Grover YP, Charan S.The authors describe a rapid and simple dot immunobinding assay (DIA) for detection and identification of equine herpesvirus-1 antigen in field samples from cases of abortion, stillbirth, perinatal foal mortality and paralysis. The assay employs a nitrocellulose membrane to which antigen is adsorbed as a dot. Antigen is identified as a coloured dot using a procedure based on the principle of enzyme-linked immunosorbent assay (ELISA). In all, 61 samples were tested by DIA and the test was compared with conventional agar gel immunodiffusion (AGID). With DIA, 44 (72%) samples gave positive result...
Duquesne F, Breuil MF, Hans A, Petry S.The cultural diagnosis of the causal agent of contagious equine metritis (Taylorella equigenitalis) using transport swabs is challenging. Swabs must be placed in Amies charcoal medium, refrigerated during transport, and plated out at the laboratory no later than 48 h after sampling. In this study, the viability of T. equigenitalis strain CIP 79.7T in 11 commercial swab transport systems was initially compared at 1 day and 2 days of storage at ambient (20 ± 3 °C) or refrigerated (5 ± 3 °C) temperature. The four best swab transport systems, systems B, E, F (used as the reference) and K, were...
Huhtinen M, Peippo J, Bredbacka P.Embryo biopsy has been used to detect inherited disorders and to improve the phenotype by analyzing of linkages between marker loci and the desired characteristics. Unfortunately, early procedures required the removal of a large portion (one-half) of the embryo for analysis, and the transfer of bisected equine embryos has not been particularly successful. Recent discovery of the polymerase chain reaction (PCR) has made possible the detection of specific DNA sequences from only a few cells. We investigated whether the removal of a small biopsy would allow for successful PCR and normal embryonic...
Evenson DP, Jost LK, Varner DD.Data from the sperm chromatin structure assay (SCSA), a flow cytometric measurement of susceptibility of sperm nuclear DNA to denaturation, show strong correlation with the fertility potential of bulls, boars, men and stallions. Previous studies showed a strong relationship between stallion spermatozoa with denatured DNA and the presence of DNA strand breaks. In the present study, the relationship between stallion sperm DNA denaturation and the redox status of -SH groups on the cysteine residues of sperm nuclear protamines that are thought to stabilize chromatin was investigated. Semen samples...
Eberhardt C, Gerhards H.The three tests (EQUI Z-Test, AGLUTINADE FOAL IMMUNITY, CITE Foal IgG-Test) were evaluated for their accuracy and usefulness in the field. Single radial immunodiffusion was used as reference method. All tests were easily and rapid to perform and results were obtained within a few minutes. It was easy to get the results of the CITE Foal IgG-Test, but use of the EQUI Z-Test and the FOAL AGLUTINADE IMMUNITY-Test needed some practice to get correct results. Results obtained by the CITE Foal IgG-Test correlated to single radial immunodiffusion in 94%, those obtained by FOAL AGLUTINADE IMMUNITY-Test...
Butudom P, Foreman JH, Kline KH, Whittem EL.Some methods of lactate (LA) measurement have not been validated appropriately for use in horses. Objective: To validate 2 LA analysers (YSI 2300 Stat Plus and TDx Lactic Acid Assay) for use with equine plasma and to compare plasma [LA] determined by the 2 methods. Methods: Both instruments were evaluated for linearity, parallelism, recovery and precision using serial dilutions of standard LA solutions and equine plasma and then comparing results with linear regression or paired t tests. Plasma [LA] results were compared in 275 blood samples collected from horses exercising at various intensit...
Dillon AM, Heath MF.Protein tyrosine phosphorylation (PTP) in thrombin- and platelet-activating factor (PAF)-stimulated equine platelet activation was investigated in the absence and presence of 2 protein tyrosine kinase inhibitors (PTKIs), methyl 2,5-dihydroxycinnamate (MDHC) and genistein. Washed equine platelets aggregated irreversibly in response to thrombin or PAF in an agonist concentration dependent fashion. MDHC produced an MDHC concentration and time dependent inhibitory effect on rate and extent of thrombin- and PAF-induced aggregations, whereas the effect of genistein on the same parameters was only ge...
Yu DT, Gale RP, Kacena A, Pearson CM.Rosette formation between human lymphocytes and horse red blood cells could be promoted by a low pH medium, overnight incubation and a temperature of 4 degrees C. The percent of sheep, horse and human rosette-forming cells in the peripheral blood were 71.7 +/- 1.8, 30.5 +/- 2.8 and 28.3 +/- 3.4 respectively. However, their percentages in thymuses were 97.1 +/- 1.1, 91.4 +/- 2.4 and 89.0 +/- 3.4. Using preparations of isolated subpopulations, it was observed that the horse and human red cell rosette-forming cells were probably also "early" sheep red cell rosette-forming cells. Rosette formation...
Horteloup MP, Threlfall WR, Funk JA.The horse early conception factor (ECF) test is designed for qualitative determination of the ECF glycoprotein in the mare that has conceived. The objectives of this study were to determine the performance of the horse ECF test for the detection of the non-pregnant mare, and to determine the agreement among subjects or "readers" regarding the interpretation of the test. Blood samples from 60 mares were collected on Days 0, 5, 8, 11 and 18 following ovulation. Pregnancy status diagnosed with the ECF test was compared (2 x 2 table) to pregnancy status diagnosed by palpation per rectum and ultras...
Nakao T, Ohno-Fujitani T, Nakao M.Approximate molecular weights and the subunit structures of Na,K-ATPase from horse kidney were estimated by means of the combination of porous silica gel chromatography, laser light scattering (LS) and refractive index (RI) measurements in C12E8. When the enzymes were eluted with NaCl- or KCl-containing solution, 3 or 4 protein peaks, respectively were detected except that of low molecular weight range. These peaks were tentatively named Na-1, Na-2, Na-2', Na-3 (NaCl-containing eluents), K-1, K-2, K-3 (KCl-containing eluents), respectively. Na,K-ATPase and K-p-nitrophenylphosphatase activities...
Levine RA, Hart AH, Wardlaw SC.Using quantitative buffy coat analysis (QBCA), rapid and accurate measurements can be made of the erythrocyte PCV, total WBC count, and platelet count, and the leukocyte population can be differentiated into total granulocytes (including quantitation of eosinophils), and lymphocytes and monocytes. The QBCA is performed by placing a blood sample (50 to 111 microliters) into a high-precision-bore microhematocrit tube that contains a freely moving, closely fitting, cylindrical plastic float. After centrifugation for 5 minutes, the buffy coat components separate by density. The plastic cylinder fl...
Reilly CA, Aust SD.An intracellular, membrane-bound enzyme exhibiting both p-phenylenediamine oxidase activity and ferrous iron oxidase activity was isolated with the plasma membrane fraction of horse heart and studied for its ability to load iron into ferritin. The ferroxidase activity of the tissue oxidase was stimulated approximately twofold by horse spleen apoferritin, and the iron was loaded into ferritin. The loading of iron into ferritin by the tissue oxidase was inhibited by anti-horse serum ceruloplasmin antibody. The stoichiometry of iron oxidation and oxygen consumption during iron loading into ferrit...
Liang CZ, Cao RB, Wei JC, Zhu LH, Chen PY.According to the antigenic analysis of equine arteritis virus (EAV) GL protein, one pair of primers were designed, with which the gene fragment coding the high antigenic domain of EAV GL protein was amplified from the EAV genome. The cloned gene was digested with BamH I and Xho I and then inserted into pET-32a and resulted pET-GL1. The pET-GL1 was transformed into the host cell BL21(DE3) and the expression was optimized including cultivation temperature and concentration of IPTG. The aim protein was highly expressed and the obtained recombinant protein manifested well reactiongenicity as was c...
McDowell KJ, Adams MH, Williams NM.Proteins synthesized and released in vitro by oviducts collected from horse mares during estrus and at day 4 after ovulation for bred and nonbred mares were examined by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS PAGE) and fluorography. Ampullary and isthmic regions both produced a wide array of nondialyzable proteins in culture. Major proteins or groups of proteins identified according to relative molecular weight (kDa) and apparent isoelectric point (pI) were at 100 kDa, pI 8; 100-200 kDa, pI 6; 150 kDa, pI 4.5; 60-100 kDa, pI 4; and an array of polypep...
Bai Y, Tong T, Liu G, Chen W, Zhang W, Wang Q, Yang T, Bu Z, Wu D.Interferon gamma (IFN-gamma) is a pleiotropic cytokine that is recognized as an important modulator of the immune response. To date, there is no report that prokaryocyte-derived recombinant equine IFN-gamma has antiviral activity. In this report, the gene coding equine IFN-gamma (EIFN-gamma) mature protein was cloned into pET-28a (+) and the recombinant EIFN-gamma was expressed in Escherichia coli (E. coli). The antiviral activity of expressed recombinant EIFN-gamma was evaluated by using a recombinant Vesicular Stomatitis Virus expressing green fluorescence protein (rVSV-GFP) system in the eq...
