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.
Love CC, Brinsko SP, Rigby SL, Thompson JA, Blanchard TL, Varner DD.The relationship between seminal plasma level (0, 10, or 20%) and extender type [Kenney type (EZ-Mixin-CST) or Kenney-modified Tyrodes-KMT] to the susceptibility of sperm DNA to denaturation and sperm motility measures were investigated in cooled (5 degrees C) stallion sperm. Three ejaculates from each of three fertile stallions were collected in an artificial vagina and processed as follows: diluted one part uncentrifuged semen with four parts of extender to a final concentration of 20% seminal plasma in either CST or KMT (20% CST; 20% KMT); diluted to a final concentration of 25 million sper...
Heidmann P, Tornquist SJ, Qu A, Cebra CK.To determine whether epsilon-aminocaproic acid (EACA) administered IV affects hemostasis and fibrinolysis in clinically normal horses and ponies. Methods: 20 clinically normal adult horses and ponies. Methods: Blood samples were collected 24 hours before (baseline) and 1 and 5 hours after i.v. administration of a low dose (30 mg/kg) or high dose (100 mg/kg) of EACA. Platelet count, fibrinogen concentration, prothrombin time, partial thromboplastin time (PTT), D-dimer concentration, alpha2-antiplasmin activity, and thrombin-antithrombin complex concentration were measured. Values at 1 and 5 hou...
Regis WC, Fattori J, Santoro MM, Jamin M, Ramos CH.The work in the literature on apomyoglobin is almost equally divided between horse and sperm whale myoglobins. The two proteins share high homology, show similar folding behavior, and it is often assumed that all folding phenomena found with one protein will also be found with the other. We report data at equilibrium showing that horse myoglobin was 2.1 kcal/mol less stable than sperm whale myoglobin at pH 5.0, and aggregated at high concentrations as measured by gel filtration and analytical ultracentrifugation experiments. The higher stability of sperm whale myoglobin was identified for both...
Archambault D, St-Louis MC, Martin S.The genome of equine arteritis virus (EAV) produces a 3' coterminal-nested set of six subgenomic (sg) viral RNAs during virus replication cycle, and each set possesses a common leader sequence of 206 nucleotides (nt) in length derived from the 5' end of the viral genome. Given the presence of the leader region within both genomic and sg mRNAs, it is likely to contain cis-acting signals that may interact with cellular or viral proteins for RNA synthesis. Gel mobility shift assays indicated that proteins in Vero cell cytoplasmic extracts formed complexes with the positive (+) and negative (-) st...
Stephano MA, Guidolin R, Higashi HG, Tambourgi DV, Sant'Anna OA.The main features associated with pit viper envenomations include the intense local lesions such as oedema, necrosis, acute renal failure and other effects. The severity of these reactions to snakebite depends on the degree of envenomation. Lachesis muta venom (LMV) has weak lethal activity, but due to the large amount often inoculated, the effects are extremely severe and demand anti-venom with a high neutralizing capacity. LMV had the lowest neutralizing antibody induction capacity in horses when compared with that of other venoms. For example, Bothrops anti-venom serum neutralizes 180 times...
Phillips T, Ferraz I, Bell S, Clegg PD, Carter SD, Mobasheri A.Glucose serves as the major energy substrate for articular chondrocytes and as the main precursor for the synthesis of extracellular matrix glycosaminoglycans in cartilage. Chondrocytes have been shown to express several glucose transporter (GLUT) isoforms including GLUT1 and GLUT3. The aim of this investigation was to determine the effects of endocrine and cytokine factors on the capacity of equine articular chondrocytes for transporting 2-deoxy-d-[2,6-3H] glucose and on the expression levels of GLUT1 and GLUT3. Chondrocytes maintained in monolayer culture were stimulated for 24 h with TNF-al...
Henderson KM, Eayrs K.To develop a means of determining pregnancy status in horses based on measuring serum oestrone sulphate (OS) concentrations using a rapid lateral flow immunoassay, and to determine the assay's effectiveness using a visual end-point. Methods: Serum samples from mares >100 days post-mating (n=701) were assayed using a nitrocellulose membrane-based lateral flow immunoassay device. The device was developed using membrane-bound 1,3,5 (10)-estratrien-3-ol-17-one conjugated to bovine serum albumin as the capture antigen, and an OS-detection monoclonal antibody coupled to colloidal gold as the visi...
Bhuyan AK, Rao DK, Prabhu NP.Proteins meet with the stipulations of Levinthal. Two test tube variants of ferrocytochrome c (ferrocyt c) whose thermodynamic stabilities are vastly different refold to the same global minimum under a given final native condition, and they do so quickly at rates that do not reflect a strong dependence on the thermodynamic driving force. The transition-state ensemble is more unfolded-like, and the folding barrier offered is energetically sizable. The experiments involve neutral- (pH 7) and alkaline ferrocyt c pH (12.7), whose aqueous stabilities are 18 (+/-0.3) and 3 (+/-0.5) kcal mol(-)(1), r...
Desantis S, Acone F, Corriero A, Deflorio M, Zubani D, Ventriglia G, Palmieri G, De Metrio G.The distribution of sialic acid residues as well as other glycosidic sugars has been investigated in the horse oviductal isthmus during anoestrus, oestrus and pregnancy by means of lectin and pre-lectin methods. Ciliated cells and non-ciliated (secretory) cells exhibited different lectin binding profiles that were found to change during the investigated stages. Ciliated cells did not show any reactivity in the basal cytoplasm, while the supra-nuclear cytoplasm displayed a few of oligosaccharides with terminal and internal alphamannose (Man) and/or alphaglucose (Glc) during oestrus and pregnanc...
Davis DG, Schaefer DM, Hinchcliff KW, Wellman ML, Willet VE, Fletcher JM.Hypogammaglobulinemia as a result of failure of transfer of passive immunity (FTPI) is an important risk factor for infectious disease in neonatal foals. The current gold standard for determining serum immunoglobulin concentrations is radial immunodiffusion (RID). The purpose of this study was to compare immunoglobulin concentrations measured by RID with those determined by an automated turbidimetric immunoassay (TIA), which has a much shorter turnaround time. Immunoglobulin concentrations were measured by both RID and TIA in serum collected from 84 neonatal foals. Sixty-seven foals had result...
Luo Y, Uboh CE, Soma LR, Guan F, Rudy JA, Tsang DS.This method describes the simultaneous separation, identification, quantification and confirmation of betamethasone (BTM) and dexamethasone (DXM) in equine plasma by liquid chromatography (LC) integrated with multidimensional tandem mass spectrometry. Analytes were directly extracted from equine plasma by methyl tert-butyl ether (MTBE). The residues were reconstituted with sample solvent. LC separation of the analytes was performed on a Hypercarb column using acetonitrile/water/formic acid (95:5:0.5, v/v/v) as the mobile phase. Sample screening, quantification and confirmation were performed i...
Albrecht RA, Kim SK, O'Callaghan DJ.The equine herpesvirus 1 (EHV-1) EICP27 protein cooperates with either the immediate-early (IE) or the EICP0 protein to synergistically trans-activate viral promoters. GST-pulldown and co-immunoprecipitation assays revealed that the EICP27 protein's cooperation with the IE or the EICP0 protein involves its physical interaction with these viral proteins. In the case of the IE-EICP27 protein interaction, IE residues 424 to 826 and EICP27 residues 41 to 206 harbor the interactive domains. Electrophoretic mobility shift assays (EMSA) suggested that the EICP27 protein is not a sequence-specific DNA...
Nishita T, Takahasi M, Kasuya T, Matsui K, Ichihara N, Murakami M, Asari M.Equine carbonic anhydrase isozymes (CA-I and CA-II) were purified from erythrocytes by several column chromatography. Polyclonal anti-CA-I and anti-CA-II sera were produced in rabbits. Sensitive competitive enzyme-linked immunosorbent assays (ELISA) were established to determine the developmental changes in CA-I and CA-II levels in equine erythrocytes. Concentrations of CA-I and CA-II in erythrocytes from 150 clinically normal thoroughbreds (123 racehorses and 27 riding horses) were determined by ELISA. Mean (+/- SD) concentrations of CA-I and CA-II in racehorses were 1.70 +/- 0.48 and 0.94 +/...
Hornyák A, Dénes B, Szeredi L, Dencsö L, Rusvai M.Two monoclonal antibodies against the Bucyrus strain of equine arteritis virus (EAV) were produced, and according to immunoperoxidase reaction following Western blot of electrophoresed EAV structural proteins, they recognized the nucleocapsid (N) protein antigen (14-kDa protein). Besides reacting with the blotted polypeptide, the antibodies of the two clones (designated 1H1 and 4G6) selected from 576 have shown high affinity and specificity to intracellular virus antigen as well. Both antibodies reacted with the representatives of the different subtypes of equine arteritis virus providing a su...
Gustafson-Seabury A, Raudsepp T, Goh G, Kata SR, Wagner ML, Tozaki T, Mickelson JR, Womack JE, Skow LC, Chowdhary BP.High-resolution gene maps of individual equine chromosomes are essential to identify genes governing traits of economic importance in the horse. In pursuit of this goal we herein report the generation of a dense map of horse chromosome 22 (ECA22) comprising 83 markers, of which 52 represent specific genes and 31 are microsatellites. The map spans 831 cR over an estimated 64 Mb of physical length of the chromosome, thus providing markers at approximately 770 kb or 10 cR intervals. Overall, the resolution of the map is to date the densest in the horse and is the highest for any of the domesticat...
Kleiber C, McGorum BC, Horohov DW, Pirie RS, Zurbriggen A, Straub R.Equine recurrent airway obstruction (RAO) is thought to result from an aberrant immune response to inhaled antigens, modulated by T lymphocytes via the secretion of pro-inflammatory cytokines. However data relating to the phenotypes of the T lymphocytes present in peripheral blood and bronchoalveolar lavage fluid of RAO horses and their cytokine profiles are contradictory. The aim of this study was to further investigate the cytokine (IL-4, IL-5, IL-13 and INF-gamma) mRNA expression profile in peripheral blood lymphocytes and bronchoalveolar lavage lymphocytes from RAO and control horses, befo...
Gutmann S, Zawatzky R, Müller M.Interferon-gamma (IFN-gamma) is a key cytokine in cell-mediated immunity. To measure IFN-gamma production of equine lymphocytes (eqIFN-gamma), we developed a quantitative ELISA. Monoclonal antibodies (mAb) were produced against bacterially derived eqIFN-gamma. The mAbs recognised recombinant and lymphocyte-derived eqIFN-gamma in ELISA, Western blotting, as well as flow cytometric and microscopic analysis. In contrast to bacterially derived material, mammalian and insect cell-derived eqIFN-gamma was biologically active but could be neutralised by one of the monoclonal antibodies. Unexpectedly, ...
Kolodziejek J, Dürrwald R, Herzog S, Ehrensperger F, Lussy H, Nowotny N.The aim of this study was to gain more detailed insights into the genetic evolution and variability of Borna disease virus (BDV). Phylogenetic analyses were performed on field viruses originating from naturally infected animals, the BDV vaccine strain 'Dessau', four widely used laboratory strains and the novel BDV subtype No/98. Four regions of the BDV genome were analysed: the complete p40, p10 and p24 genes and the 5'-untranslated region of the X/P transcript. BDV isolates from the same geographical area exhibited a clearly higher degree of identity to each other than to BDV isolates from ot...
Beutelspacher SC, Ardjomand N, Tan PH, Patton GS, Larkin DF, George AJ, McClure MO.In this study we compare the ability of self-inactivating Human Immunodeficiency Virus 1 (HIV-1) and Equine Infectious Anaemia Virus (EIAV)-based vectors to mediate gene transfer to rabbit and human corneas and to a murine corneal endothelial cell line. Both vectors were pseudotyped with vesicular stomatitis virus-G (VSV-G) envelope and contained marker transgenes under the control of an internal CMV promoter. For specificity of action, the heterologous promoter in the EIAV-vector was exchanged for an inducible E-Selectin promoter, previously shown to regulate gene-expression in a plasmid syst...
Arai K, Misumi K, Carter SD, Shinbara S, Fujiki M, Sakamoto H.Cartilage oligomeric matrix protein (COMP) is abundant within cartilage; its turnover and/or degradation have been investigated in various equine joint diseases and it has been suggested that COMP fragmentation might be useful for monitoring such conditions. Objective: To determine whether COMP metabolism is compromised in equine osteoarthritis (OA) and whether COMP degradation is a useful joint marker representing cartilage destruction. Objective: A monoclonal antibody (mAb) with a higher affinity for degraded COMP allows discrimination of diseased joints by quantifying COMP levels and fragme...
Thomas GW, Bell SC, Carter SD.Fell pony foals are affected by a congenital fatal disease that leads to profound anaemia and immunodeficiency. Previous studies comparing healthy and affected foals have shown normal T-cell populations, but a severe B-lymphopenia. Objective: To measure the levels of individual immunoglobulin subisotypes in normal and affected Fell ponies and correlate these levels with the number of peripheral B-lymphocytes. Methods: Serum levels of individual immunoglobulin subisotypes were measured by ELISA and correlated with the number of peripheral B-lymphocytes (measured by flow cytometry). Results: Aff...
Cappelli K, Porceddu A, Verini-Supplizi A, Capomaccio S, Marchis FD, Falcinelli M, Gaiti A, Silvestrelli M.The identification of differentially expressed genes is a fundamental prerequisite for understanding the molecular regulation of most physiological and pathological processes. Among the procedures employed to compare mRNA populations, those that are gel-based appear to hold great promise and are considered excellent tools for studying gene expression in species, such as the equine one, for which little genomic information is available. In the present study, we evaluated two techniques for studying mRNA profiles in horse tissue, one referred to the cDNA-amplified fragment length polymorphism (A...
Traversa D, Giangaspero A, Iorio R, Otranto D, Paoletti B, Gasser RB.Habronema microstoma and Habronema muscae (Spirurida: Habronematidae) are parasitic nematodes which infect the stomach and/or skin of equids. The accurate diagnosis of gastric habronemosis is central to studying its epidemiology, but data on its distribution and prevalence are lacking, mainly due to the limitations of clinical and coprological diagnosis in live horses. To overcome this constraint, a two-step, semi-nested PCR-based assay was validated (utilizing genetic markers in the nuclear ribosomal DNA) for the specific amplification of H. microstoma or H. muscae DNA from the faeces from ho...
Hilbe M, Guscetti F, Wunderlin S, Ehrensperger F.Equine and feline dysautonomias are characterized histopathologically by degenerating neurons with chromatolysis, pyknotic and sometimes eccentric nuclei, and loss of Nissl substance in the peripheral autonomic ganglia. Because it may be difficult to distinguish pathological from post-mortem changes in affected ganglia by histopathological examination, synaptophysin was evaluated as an immunohistochemical marker. Degenerating neurons showed strong intracytoplasmic labelling indicating abnormal accumulation of synaptophysin. It was concluded that synaptophysin immunohistochemistry is a helpful ...
Hosaka Y, Teraoka H, Yamamoto E, Ueda H, Takehana K.The aim of the present study was to clarify the presence and determine the role of apoptosis in the degenerative process of the superficial digital flexor tendon (SDFT) in the horse. Samples were obtained from normal and inflamed SDFTs of horses. To detect apoptosis and to identify apoptotic cells, the samples were subjected to immunohistochemical labelling and Western blot analysis. Although a large number of cells in degenerate areas showed positive reactions with caspase-3 and single stranded DNA antibodies, cells in normal tendon samples showed very weak reactions. Excessive apoptosis was ...
Monkos K.This paper presents the results of viscosity determinations on aqueous solutions of equine, porcine and rabbit serum albumin over a wide range of concentrations and at temperatures ranging from 5 degrees C to (42-45) degrees C. The results are compared with human and bovine serum albumin previously studied. Viscosity-temperature dependence is discussed on the basis of the modified Arrhenius formula. The effective specific volume, the activation energy and entropy of viscous flow for all investigated albumins are compared. Viscosity-concentration dependence, in turn, is discussed on the basis o...
Krishnan A, Wilson A, Sturgeon J, Siedlecki CA, Vogler EA.A systematic study of water-air (liquid-vapor, LV) interfacial tension gamma(lv) of blood plasma and serum derived from four different mammalian species (human, bovine, ovine and equine) reveals nearly identical concentration-dependence (dgamma(lv)/dlnC(B); where C(B) is plasma/serum dilution expressed in v/v concentration units). Comparison of results to a previously-published survey of purified human-blood proteins further reveals that dgamma(lv)/dlnC(B) of plasma and serum is surprisingly similar to that of purified protein constituents. It is thus concluded that any combination of blood-pr...
Rossano MG, Schott HC, Murphy AJ, Kaneene JB, Sellon DC, Hines MT, Hochstatter T, Bell JA, Mansfield LS.Equine protozoal myeloencephalitis (EPM) is a serious neurological disease of horses in Americans. Most cases are attributed to infection of the central nervous system with Sarcocystis neurona. Parasitemia has not been demonstrated in immunocompetent horses, but has been documented in one immunocompromised foal. The objective of this study was to isolate viable S. neurona from the blood of immunocompetent horses. Horses used in this study received orally administered S. neurona sporocysts (strain SN 37-R) daily for 112 days at the following doses: 100/day for 28 days, followed by 500/day for 2...
Alber J, El-Sayed A, Lämmler C, Hassan AA, Weiss R, Zschöck M.The closely related streptococcal species Streptococcus equi subsp. zooepidemicus and S. equi subsp. equi were identified by polymerase chain reaction using oligonucleotide primers designed according to species-specific parts of the superoxide dismutase A encoding gene sodA. A further differentiation of both subspecies could be performed by amplification of the genes seeH and seeI encoding the exotoxins SeeH and SeeI, respectively, which could be detected for S. equi subsp. equi but not for S. equi subsp. zooepidemicus. A further simplification of the identification and differentiation of both...
Iyer LK, Qasba PK.Alpha-lactalbumins (LAs) and c-type lysozymes (LYZs) are two classes of proteins which have a 35-40% sequence homology and share a common three dimensional fold but perform different functions. Lysozymes bind and cleave the glycosidic bond linkage in sugars, where as, alpha-lactalbumin does not bind sugar but participates in the synthesis of lactose. Alpha-lactalbumin is a metallo-protein and binds calcium, where as, only a few of the LYZs bind calcium. These proteins consist of two domains, an alpha-helical and a beta-strand domain, separated by a cleft. Calcium is bound at a loop situated at...
Lemon AV, Goddard A, Hooijberg EH.The accessibility of thromboelastography (TEG) to general practitioners is limited by short sample storage times (30 minutes) and storage temperatures (20-23°C). Objective: We aimed to evaluate the stability of canine and equine citrated blood samples when stored for extended periods of time, both at room temperature (RT) (20-23°C) and refrigerator temperature (FT) (2-7.5°C). Methods: Citrated whole blood samples from healthy dogs and horses (n = 10 for each) were stored for 30 minutes (baseline) at RT before TEG analysis. Baseline values for TEG variables R, K, α, MA, LY30, and LY60 w...
Dourine is a parasitic venereal disease of equines caused by T. equiperdum. Humoral antibodies are found in infected animals, but diagnosis of dourine must include history, clinical, and pathological findings in addition to serology. Complement Fixation Test (CFT) is the Office International des Epizooties (OIE) recommended test for international trade; however, some uninfected equines may give inconsistent or nonspecific reactions in CFT due to the anticomplementary effects of their sera. In this study an Indirect Enzyme Linked Immunosorbent Assay (iELISA) was developed. This test could be us...
Mills PC.The effects of three vehicles, phosphate-buffered saline (PBS), ethanol (50% in PBS w/w) and propylene glycol (50% in PBS w/w) on in vitro transdermal penetration of testosterone was investigated in the horse. Skin was harvested from the thorax of five Thoroughbred horses after euthanasia and stored at -20 degrees C until required. The skin was then defrosted and placed into Franz-type diffusion cells, which were maintained at approximately 32 degrees C by a water bath. Saturated solutions of testosterone, containing trace amounts of radiolabelled [14C]testosterone, in each vehicle were applie...
McMonagle EL, Taylor S, van Zuilekom H, Sanders L, Scholtes N, Keanie LJ, Hopkins CA, Logan NA, Bain D, Argyle DJ, Onions DE, Schijns VE, Nicolson L.Interleukin-12 (IL-12) is a key cytokine in the development of cell-mediated immune responses. Bioactive IL-12 is a heterodimeric cytokine composed of disulphide linked p35 and p40 subunits. The aim of this study was to verify biologically activity of the products expressed from equine interleukin-12 (IL-12) p35 and p40 cDNAs and to establish whether equine IL-12 could be expressed as a p35/p40 fusion polypeptide, as has been reported for IL-12a of several mammalian species. We report production of equine IL-12 through expression of p35 and p40 subunits in mammalian and insect cells and of a p...
Thevis M, Opfermann G, Schänzer W.Electrospray ionization (ESI) mass spectra of 19 common beta(2)-agonists were investigated in terms of fragmentation pattern and dissociation behavior of the analytes, proving the origin of fragment ions and indicating mechanisms of charge-driven and charge-remote fragmentation. Based on these data, liquid chromatographic/ESI tandem mass spectrometric (LC/ESI-MS/MS) screening and confirmation methods were developed for doping control purposes. These procedures employ established sample preparation steps including either acidic or enzymatic hydrolysis, alkaline extraction and, in the case of eq...
Rendle DI, Armstrong SK, Heller J, Hughes KJ.Point-of-care glucometry is used commonly in clinical and research settings; however, accuracy and precision of this method are concerns. The objectives of this study were to determine the accuracy of glucometry in adult horses and the precision of duplicate measurements. Blood samples were collected from 62 horses into one plain syringe, one EDTA tube and three fluoride oxalate (FO) tubes. Immediately after collection, glucose concentrations in whole blood were determined, in duplicate, by glucometry from the syringe (plain whole blood [WB] group), EDTA tube (EDTA group) and one FO tube (FO g...
Singh S, Dohre SK, Kamthan A, Pal V, Karothia BS, Singha HS, Kumar S.Glanders is a contagious and highly fatal disease of equines with zoonotic potential. It is caused by a Gram-negative, nonmotile bacterium Burkholderia mallei. Complement fixation test (CFT) is one of the most commonly used tests for diagnosis of glanders; however, it has some limitations. A recombinant-truncated Burkholderia intracellular motility A (BimA) protein-based indirect enzyme-linked immunosorbent assay (iELISA) was previously reported by us for glanders diagnosis, which has been re-optimized in this study using a panel of glanders positive (n = 75) and glanders negative (n = 227...
Rice NR, Coggins L.In the endogenous reverse transcriptase reaction, equine infectious anemia virus is able to synthesize complementary DNA (cDNA) of 8,000 nucleotides in high yield. After 2 h in 50 muM dNTP, about 2.8 mug of cDNA per mg of protein is produced, almost 30% of which is long cDNA. The system thus compares favorably with the other two well-characterized endogenous reaction systems, Moloney murine leukemia virus and avian sarcoma virus. Elongation rates of 100 to 150 nucleotides per min have been observed; these rates are comparable to those seen with purified avian myeloblastosis virus reverse trans...
Semik-Gurgul E, Ząbek T, Fornal A, Wnuk M, Pawlina-Tyszko K, Gurgul A, Klukowska-Rötzler J, Koch C, Mählmann K, Bugno-Poniewierska M.Genetic and epigenetic alterations in the equine sarcoid, a locally invasive skin tumour of equids, are still poorly characterized. Numerous studies have provided reliable evidence for the relationship between the development of cancer and the loss of function of a number of tumour suppressor genes. In the present study, we assessed methylation levels in the promoter region of SFN, S100A14 and POU2F3 genes in sarcoid samples to clarify whether DNA methylation may be associated with previously identified changes in the expression level of these genes during the course of tumour progression. Usi...
Boyer JD, Breeden DC, Brown DL.To identify compounds in Acer rubrum that cause hemolysis or oxidation of equine erythrocytes and determine whether these toxins are found in other Acer spp. Methods: Equine erythrocytes. Methods: Washed erythrocytes were incubated with extracts and fractions of Acer spp that were separated by thin layer chromatography. Methemoglobin and hemolysis were measured spectrophotometrically. Compounds within Acer spp fractions associated with cell oxidation or hemolysis were identified by gas chromatography-mass spectrometry. Results: Erythrocytes incubated separately with either A. rubrum, A. saccha...
Mendes MA, Chies JM, de Oliveira Dias AC, Filho SA, Palma MS.Most commercial recombinant proteins used as molecular biology tools, as well as many academically made preparations, are generally maintained in the presence of high glycerol concentrations after purification to maintain their biological activity. The present study shows that larger proteins containing high concentrations of glycerol are not amenable to analysis using conventional electrospray ionization mass spectrometry (ESI-MS) interfaces. In this investigation the presence of 25% (v/v) glycerol suppressed the signals of Taq DNA polymerase molecules, while 1% (v/v) glycerol suppressed the ...
Green MK, Johnston MV, Larsen BS.To elucidate the role of high mass accuracy in mass spectrometric peptide mapping and database searching, selected proteins were subjected to tryptic digestion and the resulting mixtures were analyzed by electrospray ionization on a 7 Tesla Fourier transform mass spectrometer with a mass accuracy of 1 ppm. Two extreme cases were examined in detail: equine apomyoglobin, which digested easily and gave very few spurious masses, and bovine alpha-lactalbumin, which under the conditions used, gave many spurious masses. The effectiveness of accurate mass measurements in minimizing false protein match...
Tang PW, Crone DL.A new method for hydrolyzing steroid conjugates (both sulfates and glucuronides conjugates) that is efficient, effective, and inexpensive is described. This method comprises incubation of the conjugates--after salting-out into ethyl acetate or elution from a C18 cartridge--with anhydrous methanolic hydrogen chloride (methanolysis) for 10 min. It has been successfully applied to our routine radioimmunoassay screening and GC/MS confirmation studies of steroids in prerace and postrace equine urine samples. Comparative GC/MS studies on entire (male horse) urine samples showed that methanolysis gav...
Lepczyński A, Ożgo M, Dratwa-Chałupnik A, Robak P, Pyć A, Zaborski D, Herosimczyk A.The main objectives of the study were to: (1) deeply analyse the serum protein composition of Equus caballus, (2) assess the effectiveness of the high-abundant protein depletion and improve the concentration of medium- and low-abundant proteins. The analysis were performed on the blood plasma of three healthy part-Arabian mares. The implementation of two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation - time of flight mass spectrometry allowed us to establish a horse plasma proteome map. Serum proteins were resolved at pH 4 to 7, followed by 12% SDS-PAGE. As a resul...
Szeredi L, Hornyák A, Pálfi V, Molnár T, Glávits R, Dénes B.The occurrence of equine arteritis virus (EAV) induced equine abortions was studied with different laboratory methods during a 3-year period. Tissue samples from 96 aborted equine foetuses or newborn foals were collected from 57 farms located in different parts of Hungary. Virus isolation, polymerase chain reaction (PCR), immunohistochemistry and serology were used for the detection of EAV infection. The overall seroprevalence of EAV infection in mares was 65%. EAV induced abortion was diagnosed in eight (8.3%) cases from six (10.5%) herds. Abortion was sporadic in all herds except for one, wh...
Sanmartí J, Robles-Guirado JA, Jose-Cunilleras E, Bassols A.The determination of iCa and iMg is important in veterinary medicine, but their immediate determination in whole blood is not always possible. Their stability in other sample types and the existence of interferences must be evaluated before its use. Objective: We aimed to analyze the effects of storage time on the stability of iCa, iMg, and other analytes in whole blood, plasma, and serum samples in horses and assess the interference of heparin in these measurements. Methods: Whole blood, heparin-plasma, and serum samples from 10 horses were stored at 4°C and analyzed 1, 2, 3, 4, 5, 6, 7, 8, ...
Thevis M, Opfermann G, Krug O, Schänzer W.Isotope-dilution mass spectrometry has been employed successfully in numerous fields of analytical chemistry enabling the establishment of fast and reliable procedures. In equine sports, xanthine derivatives such as caffeine and theobromine are prohibited, and doping control laboratories analyze horse urine specimens regarding these illicit performance-enhancing drugs. Theobromine has to exceed a threshold level of 2 microg/mL, hence a robust and reliable quantitation is required. Stably deuterated theobromine and caffeine were synthesized by the reaction of xanthine or theobromine with iodome...
Santos EM, Cardoso R, Souza GR, Goulart LR, Heinemann MB, Leite RC, Reis JK.Equine infectious anemia caused by equine infectious anemia virus is an important disease due to its high severity and incidence in animals. We used a phage display library to isolate peptides that can be considered potential markers for equine infectious anemia diagnosis. We selected peptides using IgG purified from a pool comprised of 20 sera from animals naturally infected with equine infectious anemia virus. The diagnostic potential of these peptides was investigated by ELISA, Western blot and dot blot with purified IgG and serum samples. Based on the results, we chose a peptide mimetic fo...
Johannisson A, Figueiredo MI, Al-Kass Z, Morrell JM.An improved fertility prediction for stallions is of importance for equine breeding. Here, we investigate the potential of a combined staining of stallion spermatozoa for superoxide and mitochondrial membrane potential (MMP) for this purpose. Semen samples were analysed immediately after arrival at the laboratory, as well as after 24 h. Superoxide was measured by MitoSOXRed, while MMP was measured with JC-1. Menadione was used to stimulate superoxide production. In addition, other parameters of sperm quality, namely motility, membrane integrity, chromatin integrity, sperm kinematics and Hoec...
Springer NL, Smith E, Brooks MB, Stokol T.To develop a flow cytometric assay to quantify platelet-derived microparticles (PMPs) in equine whole blood and plasma. Methods: Citrate-anticoagulated whole blood from 30 healthy adult horses. Methods: Platelet-poor plasma (PPP) was prepared from fresh whole blood by sequential low-speed centrifugation (twice at 2,500 × g). Samples of fresh whole blood and PPP were removed and stored at 4° and 24°C for 24 hours. Platelet-derived microparticles were characterized in fresh and stored samples on the basis of the forward scatter threshold (log forward scatter < 10(1)) and labeling with anne...
Lindeberg H, Karjalainen H, Koskinen E, Katila T.A study was performed to test a new semen collection device (Equidame phantom) that fractionates the ejaculate by comparing the quality of semen obtained by the Equidame phantom with that obtained by a Missouri artificial vagina. Semen from 4 Finnhorse stallions was collected 4 times per stallion by both methods. Half of the ejaculate was frozen and the other half extended and loaded into 2 Equitainer transport containers (24- and 48-h samples). Motility parameters were determined by a Hamilton-Thorn motility analyzer after cooled storage for 24 and 48 h and again after freezing/thawing. Raw a...
Matsumura Y, Li J, Ikeguchi M, Kihara H.Acidic buffer conditions are known to stabilize helix-rich states of even those proteins with a predominantly beta-sheet native secondary structure. Here we investigated whether such states also exist under alkaline buffer conditions. The guanidine hydrochloride (GuHCl)-induced unfolding transition and kinetic refolding of equine beta-lactoglobulin (ELG) by GuHCl-jump were investigated at pH 8.7 by far-ultraviolet circular dichroism. We found that an equilibrium intermediate appeared in 45% ethylene glycol (EGOH) buffer with 1.5 M GuHCl. The intermediate is rich in non-native alpha-helix, whic...
Miksovská J, Day JH, Larsen RW.Carbon monoxide binding to myoglobin was characterized using the photothermal beam deflection method. The volume and enthalpy changes coupled to CO dissociation were found to be 9.3+/-0.8 mL x mol(-1) and 7.4+/-2.8 kcal x mol(-1), respectively. The corresponding values observed for CO rebinding have the same magnitude but opposite sign: Delta V=-8.6+/-0.9 mL x mol(-1) and Delta H=-5.8+/-2.9 kcal x mol(-1). Ligand rebinding occurs as a single conformational step with a rate constant of 5 x 10(5) M(-1) s(-1) and with activation enthalpy of 7.1+/-0.8 kcal x mol(-1) and activation entropy of -22.4...
Cassart D, Fett T, Sarlet M, Baise E, Coignoul F, Desmecht D.The morphopathological picture of a subset of equine myopathies is compatible with a primary mitochondrial disease, but functional confirmation in vivo is still pending. The cationic dye JC-1 exhibits potential-dependent accumulation in mitochondria that is detectable by a fluorescence shift from green to orange. As a consequence, mitochondrial membrane potential can be optically measured by the orange/green fluorescence intensity ratio. A flow cytometric standardized analytic procedure of the mitochondrial function of equine peripheral blood mononuclear cells is proposed along with a critical...
Rae C, Board PG, Kuchel PW.In mammalian red blood cells the metabolism of methylglyoxal, and some alpha-ketoaldehydes, takes place via two, generally, highly active enzymes, glyoxalase 1 and 2. The 1H NMR spin-echo spectra of horse erythrocytes, and the various reactants in the glyoxalase system, were characterized as a prelude to obtaining series of spectra in time courses of methylglyoxal metabolism. We characterized the kinetics of the enzyme system in red cells from a normal horse and also from one which had very low activity of glyoxylase 2. The kinetics of the reaction scheme, with methylglyoxal as the starting su...
Al-Khaldi K, Yimer N, Al-Bulushi S, Haron AW, Hiew M, Babji AS.The aim of this study was to evaluate the effects of adding different concentrations of edible bird's nest (EBN) which is secreted by swiftlet birds (), into EquiPlus® and E-Z Mixin® extenders on the quality of chilled Arabian stallion semen at various storage times (0, 24 and 48 h). Ten ejaculates were collected from five stallions, and diluted using the two extenders containing 0% (control), 0.12%, 0.24% and 0.24% of EBN + seminal plasma (SP). All the diluted semen samples were then cooled and stored at 5 °C, and examined at 0, 24 and 48 h. Sperm kinetic parameters were assessed using com...
Kakoi H, Kijima-Suda I, Gawahara H, Kinoshita K, Tozaki T, Hirota K, Yoshizawa M.To construct a system for identifying individual horses from urine samples that are submitted for postracing doping tests, we developed a genotyping assay based on 26-plex single-nucleotide polymorphisms (SNPs). DNA was isolated from urine using a commercially available DNA/RNA extraction kit, and SNP genotyping was achieved with a SNaPshot(™) technique. DNA profiles including 26 SNPs were acquired from urine samples and blood/hair samples. Within the studied Thoroughbred population, the 26-plex assay showed a probability of identity of 5.80 × 10(-11). Compared to the conventional short tan...
McFadden AM, Pearce PV, Orr D, Nicoll K, Rawdon TG, Pharo H, Stone M.To summarise investigation and laboratory data collected between 2001 and 2011 to provide evidence that equine arteritis virus is not present in the horse population of New Zealand. Methods: Analysis was carried out on results from laboratory tests carried out at the Ministry for Primary Industries Animal Health Laboratory (AHL) for equine arteritis virus from horses tested prior to being imported or exported, testing of stallions as part of the New Zealand equine viral arteritis (EVA) control scheme and testing as part of transboundary animal disease (TAD) investigations for exclusion of EVA....
Slusarewicz P, Slusarewicz JH, Nielsen MK.An automated equine fecal egg count test, known as the Parasight System, was modified for use with small ruminants. Modifications included the introduction of a short centrifugation step in a floatation medium, an adjustment in pre-test sample filtering, and training of an image analysis-based egg counting algorithm to recognize and enumerate trichostrongylid eggs. In preliminary assessments, the modified method produced trichostrongylid egg counts comparable to manual McMaster analyses of the same samples from both ovine and caprine sources. The coefficient of determination (R) for the linear...
