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.
Morris DD, Moore JN, Fischer K, Tarleton RL.A study was performed to determine whether equine macrophages produce tumor necrosis factor (TNF) activity in vitro in response to endotoxin and to study the effects of endotoxin concentration and incubation time on the amount of TNF produced. Equine peritoneal macrophages were isolated and cultured in vitro for 2, 6, 12, or 24 hr in tissue culture media containing 1) no additive (nonstimulated control), 2) endotoxin (0.5 ng/ml, 5 ng/ml, or 5 micrograms/ml), or 3) the calcium ionophore A23187 (0.95 microM). The supernatant media concentrations of TNF activity were determined by an in vitro cyt...
Egberink HF, Ederveen J, Montelaro RC, Pedersen NC, Horzinek MC, Koolen MJ.Feline immunodeficiency virus (FIV) grown in cat lymphocyte and thymocyte cultures was labelled with L-[35S]methionine or [3H]glucosamine and virus-coded proteins were identified using immunoprecipitation. Polypeptides with apparent Mr values of 15K, 24K, 43K, 50K, 120K and 160K were detected. An additional polypeptide of 10K was detected by Western blot analysis. The two highest Mr species sometimes appeared as one band, of which only the 120K polypeptide was glycosylated. In the presence of tunicamycin gp120 was no longer detectable and a non-glycosylated precursor of 75K was found instead. ...
Auer DE, Ng JC, Seawright AA.Polymorphonuclear leukocytes (PMNLs) were isolated from an inflammatory exudate induced in the intercarpal joints of horses by an administration of carrageenin. Their superoxide production at rest and following stimulation with either serum-treated zymosan (STZ) or phorbol myristate acetate (PMA) was measured by cytochrome-c reduction. Stimulation of the cells increased the cytochrome-c reduction 10-15 times that of resting cells. The maxima were 20 nmol of reduced cytochrome-c per 10(6) cells per ml at 120 min (STZ) and 35 nmol of reduced cytochrome-c per 10(6) cells per ml at 60 min (PMA). T...
May SA, Hooke RE, Lees P.Equine interleukin-1 has been produced from peripheral blood monocytes by stimulation with E. coli lipopolysaccharide. Sephacryl S200 gel filtration revealed a molecular weight of 17-18 kD. Chromatofocusing of the 17-18 kD peak identified four active fractions. Two major peaks were detected at pH 6.7 and pH 7, with smaller peaks at pH 6.3 and pH 5.9. The pI 7 molecule is probably the equine form of IL-1 beta.
Mollerach-Gobbi B, Retegui LA, Peña C.The immunochemical behavior of several fragments of equine growth hormone (eGH) was examined using competitive binding assays with antibodies (Abs) to eGH obtained from different sources. Antigenicity was detected within the sequences 5-72 and 73-123 by rabbit Abs to eGH and by three mouse monoclonal antibodies (MAbs) produced by using bovine growth hormone as immunogen, but showing heteroclitic properties towards eGH. The polyclonal Abs to eGH also recognized as immunoreactive two smaller peptides corresponding to the amino acid residues 52-72 and 110-123. By contrast, the heteroclitic Abs to...
Ekfalck A.A method was developed for separating different layers of the matrix of the equine hoof wall by dissection, and the layers were then analyzed with respect to their amino acid composition. The results were used to compare the biochemistry of hard keratinization (e.g., in the hoof wall matrix) and soft keratinization (e.g., in the epidermis of the skin). Hard keratinization differed from soft keratinization not only by its previously well known high incorporation of cystine, but also by considerable incorporation of tyrosine and threonine into the outer layers of the keratogenous zone and by the...
Baker TS, Newcomb WW, Booy FP, Brown JC, Steven AC.Cryoelectron microscopy and three-dimensional computer reconstruction techniques have been used to compare the structures of two types of DNA-free capsids of equine herpesvirus 1 at a resolution of 4.5 nm. "Light" capsids are abortive, whereas "intermediate" capsids are related to maturable intracellular precursors. Their T = 16 icosahedral outer shells, approximately 125 nm in diameter, are indistinguishable and may be described in terms of three layers of density, totalling 15 nm in thickness. The outermost layer consists of protruding portions of both the hexon and the penton capsomers, ris...
Shams el Din HE, el Nasri M.Seventeen isolates (4.27%) were recovered from 398 samples. Twelve isolates (4%) were obtained from 300 donkey nasal swabs, three (4.3%) and two (6.89%) isolates were recovered from 69 horse nasal swabs and 29 mare uterine washings, respectively. Nine isolates were lost during storage at -20 degrees C and the remaining eight were identified as mycoplasmas and their biological, biochemical and serological reactions were investigated. The isolates could be divided into two groups on the basis of glucose fermentation and arginine hydrolysis. The first group neither fermented glucose nor hydrolyse...
Adam A, Ong H, Sondag D, Rapaille A, Marleau S, Bellemare M, Raymond P, Giroux D, Loo JK, Beaulieu N.A monoclonal antibody was synthesized in mouse against the O-(3-carboxypropionyl) derivative of albuterol linked to bovine serum albumin. Isotyping of this material revealed the IgG1 class characterized by an affinity constant of 1.03 nM-1 and a density of sites of 0.55 nM. This antibody was found specific as its cross-reactivity to structurally related molecules was less than 1% except for clenbuterol (75%). A radioimmunoassay was set up with culture supernatant (final dilution 1/1000) and [3H] albuterol. The calibration curve was characterized by a maximum binding of 28%, an ED50 of 1.15 pmo...
Schmid DO, Ek N, Braend M.A silent allele in the transferrin locus (Tf) was observed in a Thoroughbred mare and in five of her offspring from three different matings. Evidence for the silent allele was obtained by quantitative immunodiffusion studies.
Bowling AT, Dileanis S.The C3 polymorphism of equine serum or plasma revealed by agarose gel electrophoresis can be diagnosed with protein stain following acid protein fixation. In addition to the three alleles previously described (C31, C32, C33), a fourth allele (C34) was found. Population data for 25 domestic breeds and Equus przewalskii are presented.
Milne EM.Equine serum haptoglobin was separated by polyacrylamide gel isoelectric focusing and visualized by protein staining or Western blotting. Conventional protein staining revealed up to three bands in the pI range 4.17 to 4.44. The blotting technique, however, showed an anodal group of 8 to 10 bands with a pI range of 4.11 to 4.52 and a cathodal group of 4 to 6 bands with a range of 4.55 to 5.14. The blotting method revealed that equine haptoglobin migrates outside the prealbumin area, in contrast to previous reports.
Franke D, Maasch HJ, Wahl R, Schultze-Werninghaus G, Bretting H.We investigated five horse epithelial allergen extracts prepared from different qualities of raw material by several biochemical and immunochemical methods. Horse serum albumin and horse serum were used to identify serum-related antigens. We found high similarities as well as marked differences between the extracts. There were strong differences in the protein contents, the protein patterns obtained by isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the total allergenic activities obtained by radioallergosorbent test inhibition assays and the amounts and num...
Rivero JL, Cascone O, Biscoglio de Jimenez Bonino MJ.1. The method of Kubota et al. [Biochim. biophys. Acta 701, 242-252 (1982)] was applied to several members of the growth hormone family in order to examine their conformational homology. 2. The method neither detects differences between rat, cow, sheep, horse and alpaca hormones, nor between monkey and human hormones. 3. Lack of homology between primate and non-primate growth hormones was found in segments 42-49 and 184-191. The first fragment could be linked to species-specificity.
Harkness RA, McCreanor GM, Allsop J, Snow DH, Harris RC, Rossdale PO, Ousey JC.1. Plasma hypoxanthine and xanthine concentrations are very low in the horse and low in rat, mouse and greyhound compared to concentrations in beagles, man, sheep and rabbit. 2. Activities in erythrocytes of the main enzyme metabolizing hypoxanthine, hypoxanthine phosphori-bosyltransferase, show a similar pattern (Tax et al., 1976, Comp. Biochem. Physiol. 54B, 209-212); thus low activities have been found where plasma concentrations were low. 3. Hypoxanthine phosphoribosyltransferase activities in horse tissue other than erythrocytes are similar to those in man and rabbit with high activities ...
Ellison RS, Jacobs RM.The main purpose of this study was to ascertain whether isoelectric point determination of alkaline phosphatase (AP) using an isoelectric focusing technique on agarose gels could define the isoenzymes present in healthy equine serum. The isoelectric points of AP extracted from nine tissues ranged from pH 3.5 to 7.5 with all tissues having multiple bands. There was considerable similarity in band pattern among tissues, with only pancreatic and colostral AP having substantially different isoelectric points from the others. Sera contained thirteen bands with isoelectric points ranging from pH 3.5...
House C, Mikiciuk PE, Berninger ML.Five serological methods of diagnosing African horse sickness were evaluated, using a battery of serum samples from experimental horses vaccinated and challenged with each serotype of African horse sickness virus (AHSV1 through AHSV9): agar gel immunodiffusion (AGID), indirect fluorescent antibody (IFA), complement fixation (CF), virus neutralization (VN), and enzyme-linked immunosorbent assay (ELISA). The 5 tests were also compared using a panel of field samples, convalescent equine sera with antibodies to domestic equine viral diseases, and sera from horses awaiting export. The ELISA describ...
Patterson SD, Bell K, Manton VJ.A detailed biochemical characterization of four of the five previously described alleles of the plasma protease inhibitor (Pi) system of Equus przewalskii was performed using both one- and two-dimensional electrophoretic techniques. The proteins have been characterized in terms of isoelectric point, relative molecular mass, inhibitory activity to bovine trypsin and chymotrypsin, immunochemical cross-reactivity, terminal sialic acid content and enzyme:inhibitor complex formation and the oxidation sensitivity of this interaction. Using these functional criteria, only three loci (Spi 1, 2 and 3) ...
van den Heuvel LP, van den Born J, Veerkamp JH, Janssen GH, van de Velden TJ, Monnens LA, Schröder CH, Berden JH.1. Proteoglycans extracted from human and equine glomerular basement membranes (GBM) were purified by ion-exchange chromatography and gel filtration. 2. The glycoconjugates had an apparent molecular mass of 200-400 kDa and consisted of 75% protein and 25% glycosaminoglycan. Glycosidase and HNO2 treatment and the amino sugar and sulfate composition of both proteoglycan preparations identified heparan sulfate (HS) as the predominant saccharide chain. 3. Hydrolysis with trifluoromethanesulfonic acid yielded comparable core proteins with molecular masses of ca 160 and 120 kDa. 4. The HS chains had...
Rwambo PM, Issel CJ, Hussain KA, Montelaro RC.A neutralization escape mutant (A/1 E) of equine infectious anemia virus was isolated after 13 passages in cell culture in the presence of serum containing antibodies to type- and group-specific determinants of EIAV envelope glycoproteins. Loss of neutralization by the selecting serum correlated with loss of two epitopes in the major envelope glycoprotein gp90 of A/1 E which were present in a parallel variant isolated from a persistently infected pony.
Martin B, Silberzahn P.A significant decrease of CBG binding capacity in plasma of the mare throughout pregnancy was demonstrated using equilibrium dialysis and gel equilibration methods. As indicated with immunoelectrophoresis experiments, the pregnancy related fall of CBG binding capacity was linked to an actual decrease in blood CBG concentration. This result contrasts sharply with data on most other mammalian species, with the exception of the gestating rhesus monkey.
Koskinen E, Lindeberg H, Kuntsi H, Katila T.Twenty-four Finnhorse mares were examined by rectal palpation and ultrasonography every 6 h during late oestrus to determine the time of ovulation. Milk and serum samples were collected every 6 h after the detected ovulation for progesterone analysis. The progesterone rises took place within 0-54 h and 0-60 h after ovulation, in milk and serum, respectively. Statistically significant differences (p less than 0.05) in progesterone levels were observed for the first time 12-18 h and 18-24 h after ovulation, in serum and milk, respectively, as compared to progesterone levels 0-6 h after ovulation...
Bowling AT, Gordon L, Penedo MC, Wictum E, Beebout J.We describe a method for agarose IEF under acid conditions in which a single gel can be used to diagnose from equine red cell lysates genetic variants for carbonic anhydrase (CA) and catalase (Cat). Family and population data for 4801 horses of 27 breeds and seven trap sites of Great Basin feral horses are presented to support the presence of a sixth CA allele, CAE, which has been recognized previously, but not described by published data. Allelic frequencies for the two systems suggest it may be appropriate to use this gel for parentage verification programmes or to obtain population data for...
Meijer AE, van den Hoven R.In this communication, the results of a histochemical and biochemical enzyme study on gluteus medius muscle of horses, sensitive to exertional myopathy, during attacks of rhabdomyolysis are presented. For the biochemical study the biopsy specimens investigated were selected by means of histological and enzyme histochemical staining methods. Dissected specimens were used which contained groups of muscle fibres with a high or low activity of glucose-6-phosphate dehydrogenase. The activity of glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, glutathione reductase, glutathione per...
López-Rivero JL, Agüera E, RodrÃguez-Barbudo MV, Galisteo AM, Morales-López JL.Samples taken from the middle gluteal muscle of 95 untrained adult horses of different ages and sex were subjected to histochemical analysis using the myosin adenosine triphosphatase (m-ATPase) and nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) staining techniques. Fibres were classified into types I, IIA and IIB according to m-ATPase activity after preincubation at pH 4.4. The percentage of FT (Fast-Twitch Glycolytic) fibres and the proportion of IIB fibres with "high" and "low" oxidative capacity were determined in serial sections stained for NADH-TR. Statistical analysis ...
Patterson SD, Bell K.The carbohydrate side chains of the major plasma serpins of the horse and wallaby have been characterized by lectin analyses of protein blots from two-dimensional gels using the major human plasma serpin, alpha 1-protease inhibitor, as a control. Eight lectins were used in the characterization in conjunction with enzymatic deglycosylation of complex and high mannose side chains, chemical desialylation and defucosylation, and one round of 'Smith degradation', all being performed on the nitrocellulose blots. Assuming a standard complex side chain structure, the results of the 21 lectin/treatment...
Welsch U, Schumacher U, Buchheim W, Schinko I, Jenness P, Patton S.A specific secretory product of the lactating mammary gland are triglyceride fat globules which are enveloped by a very complex membrane, the milk fat globule membrane (MFGM). In different mammalian species (man, rhesus monkey, horse, goat, sheep, cow, grey seal, camel, alpaka) the glycoproteins of this membrane have been analyzed by gel electrophoresis, Western blotting and lectin histochemistry. A remarkable intra- and interspecific variability of these glycoproteins has been detected pointing to so far unknown physiological adaptions, which may play a role in the intestine of the new born. ...
Williams RC.The evolutionary distance between two sets of proteins was estimated using the techniques of Miyata and Yasunaga (1980) and Kimura (1980). Human beta 2-microglobulin was compared with the homologous murine molecule, while human and equine alpha-globin were similarly treated. It was found that a large amount of molecular evolution has occurred in beta 2-microglobulin since its divergence from the common ancestor of mice and humans. Kimura's estimate of evolutionary distance, K, is 0.353, while those of Miyata and Yasunaga are KS = 0.708 and KA = 0.171. The respective values for human and equine...
Henderson K, Stewart J.A dipstick, competitive immunoassay for rapidly measuring serum oestrone sulfate (OS) concentrations in horses was developed to distinguish mares 100 or more days pregnant from non-pregnant animals. 6-Ketoestrone 6-carboxymethyloxime conjugated to bovine serum albumin (oestrone CMO-BSA) was 'dotted' 25 mm from the bottom edge of 45 x 5 mm strips of polyester-film-supported cellulose nitrate membrane, pore size 3 microm. The strips were blocked, dried and a 15 x 5-mm cellulose absorbent sink attached 10 mm from the top of each strip. The manufactured dipsticks were stored with desiccant at room...
Yamaguchi K, Sawada T, Yamane S, Haga S, Ikeda K, Igata-Yi R, Yoshiki K, Matsuoka M, Okabe H, Horii Y, Nawa Y, Waltrip RW, Carbone KM.Borna disease virus (BDV) is a neurotropic pathogen that infects a wide variety of vertebrates. We have developed a new electrochemiluminescence immunoassay (ECLIA) for the detection of antibodies to BDV, using three synthetic peptides corresponding to the amino acid residues 3-20 and 338-358 of p40 and 59-79 of p24 peptide of BDV. Using the ECLIA, we examined serum samples for the presence of anti-BDV antibodies in 20 rats (experimentally BDV-infected and uninfected) and 38 horses (13 US horses, experimentally infected and uninfected, and 25 Japanese horses, feral and domestic). The ECLIA, pe...
López-Rivero JL, Agüera E, RodrÃguez-Barbudo MV, Galisteo AM, Morales-López JL.Samples taken from the middle gluteal muscle of 95 untrained adult horses of different ages and sex were subjected to histochemical analysis using the myosin adenosine triphosphatase (m-ATPase) and nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) staining techniques. Fibres were classified into types I, IIA and IIB according to m-ATPase activity after preincubation at pH 4.4. The percentage of FT (Fast-Twitch Glycolytic) fibres and the proportion of IIB fibres with "high" and "low" oxidative capacity were determined in serial sections stained for NADH-TR. Statistical analysis ...
Megerssa YC, Gari FR, Woldemariyam FT.Validation of a test method is critical for confirming that the test can generate accurate and precise data. Although commercial biochemical test kits exist there are no specific and validated commercial clinical chemistry test kits designed for horses. The aim of this study was to validate commercial clinical chemistry test kits designed for a human serum for use in horses. Results: Blood samples were collected from 29 apparently healthy adult male horses and pooled serum was prepared. Validation comprises replication and recovery experiments. Total observable error (TE), sigma (σ) metrics, ...
Scudamore CL, Pemberton AD, Miller HR, McDonnell AM, Thomson SR, Dawson A, Watson ED.An enzyme linked immunosorbent assay (ELISA) was developed and used to estimate the concentrations of the serine proteinase inhibitor, alpha-1 proteinase inhibitor (API), in uterine flushings recovered from mares at different stages of the oestrous cycle and before and after the induction of experimental endometritis. There was a significant increase in the concentrations of API and albumin relative to total protein in flushings recovered during oestrus compared with dioestrus but no difference was observed in the concentrations of these proteins relative to total protein before and after the ...
Richards SL, Cawley AT, Raftery MJ.Dermorphin, a hepta-peptide with potent analgesic properties, is classified as a doping agent in equine racing. Since its discovery, a number of biologically active structural analogs have been synthesized and made commercially available so there is a need for reliable methods of detection. Results: A sensitive detection method was developed for dermorphin and six analogs in equine urine. Peptide enrichment was achieved using weak cation exchange with subsequent separation and detection by nano-UHPLC-MS/MS. Method validation parameters included: specificity, linearity (5-10000 pg/ml), recovery...
Hunyadi L, Datta P, Rewers-Felkins K, Sundman E, Hale T, Fajt V, Wagner S.The objective of this study was to determine the pharmacokinetic parameters of oclacitinib maleate as a top dress given to adult horses. Six adult horses with a mean weight of 528Â kg were administered a single dose of 0.5Â mg/kg oclacitinib maleate. Blood was collected prior to drug administration and at 15Â min, 30Â min, 45Â min, 1, 2, 4, 6, 8, 12, 24, 48, and 72Â h after treatment. Oclacitinib maleate plasma concentrations were measured by liquid chromatography/mass spectrometry. Pharmacokinetic parameters were found best to fit a one-compartment model. Mean C was 486Â ng/ml (range 423-549Â...
Wertz PW, Downing DT.The lipids of horse hoof have been analyzed by quantitative thin-layer chromatography. The major components include cholesterol (37-40%), six groups of ceramides (10-15%), and cholesteryl sulfate (15-20%). Free fatty acids are abundant (15.8%) in the outer fully keratinized hoof, but are present at only low levels (3.1%) in the softer hyponychium. The material identified as cholesteryl sulfate was isolated by preparative thin-layer chromatography and characterized by a combination of chemical, chromatographic, and spectroscopic methods. The infrared spectrum of the isolated material had absorp...
You Y, Uboh CE, Soma LR, Guan F, Li X, Rudy JA, Chen J.A rapid and sensitive method was developed for the screening, quantification and confirmation of ethyl glucuronide (EG) and ethyl sulfate (ES) as biomarkers for alcohol administration to racehorses using liquid chromatography coupled on-line with triple quadrupole tandem mass spectrometry. Urine sample aliquots (0.1 mL) were pre-treated by protein precipitation. Separation of EG and ES was achieved on an Ultra PFP column. Isocratic elution with a flush step was performed using 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Analysis was performed by negative electrospra...
Eckersall PD, Beeley JG, Snow DH, Thomas A.The two major polypeptides H (Mr 49,000) and L (Mr 33,000) of equine sweat have been purified by gel filtration and characterised by gel electrophoresis and compositional analysis. Both H and L are glycoproteins containing sialic acid, neutral sugars, N-acetylglucosamine and N-acetylgalactosamine, but the two polypeptides differ considerably in the extent of glycosylation. H and L also differ in amino acid composition, but both contain only low levels of sulphur containing amino acids and histidine. These glycoproteins may behave as surfactants.
Siwińska N, Żak A, Słowikowska M, Kubiak K, Jaworski Z, Niedzwiedź A.Hair is a more biologically stable material than other tissues and contains a relatively constant level of minerals, drugs or toxins. The content of essential elements in hair depends on dietary and non-dietary factors. The aim of this study was to assess and compare the effect of diet on the elemental composition and morphological properties of the hair of healthy Polish Konik horses. Mane hair was collected from 19 horses living in The National Park in Popielno in Poland. Six of these horses were free-ranged all year with permanent access to pasture, while 13 horses were kept in the stable a...
Reubel GH, Studdert MJ.Based on sequence homology with human adenovirus 2 (HAdV2), the hexon gene of equine adenovirus 1 (EAdV1) was identified. HindIII restriction fragments containing the hexon and other viral genes were cloned into the plasmids pUC19 and pBlueScript SK(-) and sequenced. The nucleotide sequence of the hexon gene was completely determined and partial sequence data were obtained for seven other EAdV1 genes. Amino acid (aa) sequence comparison with published adenovirus (AdV) proteins identified the genes for the IIIa, penton, pVII, PVI, 23K proteinase, DNA binding and 100K proteins. The eight EAdV1 g...
Patterson SD, Bell K.The carbohydrate side chains of the major plasma serpins of the horse and wallaby have been characterized by lectin analyses of protein blots from two-dimensional gels using the major human plasma serpin, alpha 1-protease inhibitor, as a control. Eight lectins were used in the characterization in conjunction with enzymatic deglycosylation of complex and high mannose side chains, chemical desialylation and defucosylation, and one round of 'Smith degradation', all being performed on the nitrocellulose blots. Assuming a standard complex side chain structure, the results of the 21 lectin/treatment...
Tetens J, Venugopal CS, Holmes EP, Koch CE, Hosgood G, Moore RM.To evaluate the in vitro effects of adenosine tryphosphate (ATP) on vasomotor tone of equine colonic vasculature. Methods: Arteries and veins from the left ventral colon of 14 mixed-breed horses euthanatized for reasons unrelated to cardiovascular or gastrointestinal tract disease. Methods: Endothelium-intact and -denuded arterial and venous rings were precontracted with 10(-7) and 1.8 x 10(-8) M endothelin-1, respectively. In 1 trial, endothelium-intact rings were also incubated with 10(-4) M N omega-nitro-L-arginine methyl ester (L-NAME) to inhibit nitric oxide (NO) production. Adenosine tri...
Goehring LS, Kessels BG, van Maanen C, Voorbij HA, Sloet van Oldruitenborgh-Oosterbaan MM.The measurement of albumin concentrations in cerebrospinal fluid (CSF) and serum for albumin quotient (AQ) calculations in normal horses was performed by 2 methods: 1) total protein measurement, followed by electrophoresis of the samples to obtain an albumin percentage; and 2) albumin immunoprecipitation quantitated by nephelometry. The results of both methods correlated well, and nephelometry was chosen to determine the albumin concentrations in CSF samples obtained from an indwelling subarachnoidal catheter for daily sampling. Because the use of an indwelling catheter to collect repetitive C...
Dunnett M, Richardson DW, Lees P.Hair analysis to detect drug administration has not been studied extensively in horses. This study aimed to (a) develop an analytical method for enrofloxacin and its metabolite ciprofloxacin in mane and tail hair, (b) relate measured values to doses, routes of administration, hair colour, and (c) demonstrate long-term detectability. Samples were extracted in trifluoroacetic acid at 70 degrees C. Extracts were cleaned-up by solid-phase extraction and analysed by high-performance liquid chromatography with UV-diode array detection. Analyte recoveries were > 87%. Horses were sampled after therape...
Mancheño JM, Jayne S, Kerfelec B, Chapus C, Crenon I, Hermoso JA.Horse pancreatic lipase-related proteins PLRP1 and PLRP2 are produced by the pancreas together with pancreatic lipase (PL). Sequence-comparison analyses reveal that the three proteins possess the same two-domain organization: an N-terminal catalytic domain and a C-terminal domain, which in PL is involved in colipase binding. Nevertheless, despite the high level of sequence identity found, they exhibit distinct enzymatic properties. The intrinsic sensitivity of the peptide bond between Ser245 and Thr246 within the flap region of PLRP2 to proteolytic cleavage probably complicates PLRP2 crystalli...
Bailly-Chouriberry L, Garcia P, Cormant F, Loup B, Popot MA, Bonnaire Y.Cobra (Naja naja kaouthia) venom contains a toxin called α-cobratoxin (α-Cbtx) containing 71 amino acids (MW 7821 Da) with a reported analgesic power greater than morphine. In 2013, the first analytical method for the detection of α-Cbtx in equine plasma was developed by Bailly-Chouriberry et al, allowing the confirmation of the presence of α-Cbtx at low concentrations (1-5 ng/mL or 130-640 fmol/mL) in plasma samples. To increase the method sensitivity and therefore to improve the detection of α-Cbtx in post-administration plasma samples, a nano-liquid chromatography-mass spectrometry/hi...
Sandbaumhüter FA, Theurillat R, Bettschart-Wolfensberger R, Thormann W.The combination of ketamine and an α -receptor agonist is often used in veterinary medicine. Four different α -receptor agonists, medetomidine, detomidine, xylazine, and romifidine, which differ in their chemical structure and thus in selectivity for the α -receptor and in the sedative and analgesic potency, are typically employed during surgery of equines. Recovery following anesthesia with ketamine and an α -receptor agonist is dependent on the α -receptor agonist. This prompted us to investigate (i) the inhibition characteristics for the N-demethylation of ketamine to norketamine and (...
Hu WG, Alvi AZ, Fulton RE, Suresh MR, Nagata LP.A recombinant gene encoding a single-chain variable fragment (scFv) antibody against Venezuelan equine encephalitis virus (VEE) was cloned into a prokaryotic T7 RNA polymerase-regulated expression vector. A streptavidin-binding peptide gene fused to a 6His tag was attached downstream to the scFv gene. The recombinant fusion protein was expressed in bacteria as inclusion bodies that were subsequently solubilized with 8 M urea and renatured by an arginine system. Purification of the fusion protein was achieved by immobilized metal affinity chromatography. Enzyme-linked immunosorbent assay (ELISA...
Naef A, Damerau B, Keller HU.The relationship between the short transient intracellular increase in cAMP levels on the one hand and chemotaxis or crawling movements on the other hand was investigated using human and equine granulocytes. C5ades arg, f-met-leu-phe, human serum albumin and immunoglobulin were used as stimulating agents. There was no strict correlation between the induction of crawling movements or of chemokinesis in general and the generation of the cAMP peak. But there was so far a strict parallelism between the occurrence of the chemotactic response and the cAMP peak. However, the magnitude of the peak was...
Mawhinney I, Davis N, Carson T, Torrens N, Wales A.The study examined and compared the sensitivity of culture and a quantitative PCR assay for screening equine semen for the presence of Taylorella equigenitalis (CEMO). Chilled semen samples, both raw and treated with extender, from two stallions were spiked with the organism at seven or 23 days postejaculation and prepared in serial dilutions. Culture of the 7-day raw semen readily detected CEMO at all dilutions, but extended semen yielded counts that were two log cycles lower at equivalent dilutions, with the organism being nearly undetectable at the maximal dilutions. By contrast, PCR sensit...
Sayers DE, Theil EC, Rennick FJ.Cell-specific variations in apoferritin structure correlate with variations in iron metabolism that suggest functional specificity of the protein shell. Using EPR spectroscopy, we previously showed that vanadyl binds to specific sites on apoferritin, and that VO2+ binding is reduced by Fe(II) and Fe(III) (the natural substrates) and by metals known to influence iron storage (Chasteen, N. D., and Theil, E. C. (1982) J. Biol. Chem. 257, 7672-7677). Such observations suggest that the metal-binding site is important to apoferritin function and may define a location where the influence of cell-spec...
Bakala A, Karlik W, Wiechetek M.In this study a detailed description of the equine hepatocyte isolation procedure is presented. Livers were obtained from horses slaughtered at the local slaughterhouse. For blood removal and liver preservation the following steps are suggested: perfusion with the oxygenated HBSS (0-2 degrees C, with continuous flow of 500-800 ml/min for 3-6 min), protection from ischemia injury by flushing with ice-cold University of Wisconsin Solution (UW, flow rate of 500-800 ml/min), and finally immersion of the liver lobe in UW solution (2 degrees C) during its transport to the laboratory. For equine isol...
Agnihotri K, Pease B, Oakey J, Campbell G.In 2011, a 2-year-old horse in northern Queensland, Australia, was reported to have developed mild neurologic signs, and a blood sample was submitted for laboratory investigation. Virus isolation was performed using the blood sample, and an orbivirus was isolated. This was confirmed to be a strain of Elsey virus (ELSV) after transmission electron microscopy and nucleotide sequencing. The nucleotide sequence was compared with those in GenBank, and had 100% identity with ELSV previously reported from the Northern Territory, Australia. ELSV is taxonomically closely related to Peruvian horse sickn...
Hermanson JW, Hegemann-Monachelli MT, Daaod MJ, LaFramboise WA.The biceps brachii of horses is subdivided into a lateral and medial head. Electrophoresis of samples from the lateral head revealed three slow-migrating native myosin isoforms, including one that does not correspond to slow myosin isoforms described for other mammalian muscles. In contrast, the medial head contained a single slow isoform. Both the lateral and medial heads contained three fast-migrating isoforms corresponding with the FM-2, FM-3 and FM-4 isoforms reported for other mammalian fast-twitch muscle fibers. Electrophoresis of myosin heavy chains (MHCs) revealed only two MHC bands, o...
Waelchli RO, Jaworski T, Ruddock WD, Betteridge KJ.Equine blastocyst fluid has a lower [Na+], a higher [K+] and a lower osmolality than does normal blood serum. Based on the assumptions that the sodium pump is primarily responsible for fluid accumulation and that ions transported actively into the blastocyst increase blastocyst osmolality above that of the external medium, we hypothesized that the [Na+] and the osmolality of mare uterine fluid are lower than those of blastocyst fluid. Microdialysis and ion chromatography were used to estimate [Na+] and [K+] of uterine fluid. Mares (n=10) were used for in vivo measurements at different stages o...
Haikerwal A, Barrera MD, Bhalla N, Zhou W, Boghdeh N, Anderson C, Alem F, Narayanan A.Acutely infectious new world alphaviruses such as Venezuelan Equine Encephalitis Virus (VEEV) pose important challenges to the human population due to a lack of effective therapeutic intervention strategies. Small interfering RNAs that can selectively target the viral genome (vsiRNAs) has been observed to offer survival advantages in several in vitro and in vivo models of acute virus infections, including alphaviruses such as Chikungunya virus and filoviruses such as Ebola virus. In this study, novel vsiRNAs that targeted conserved regions in the nonstructural and structural genes of the VEEV ...
