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.
Qin Z, Durand LG, Allard L, Cloutier G.In most studies that were aimed at evaluating the kinetics of red blood cell (RBC) aggregation, human blood was initially circulated at a high shear rate to disrupt the aggregates, and measurements were performed following a complete flow stoppage, during the process of rouleau formation. However, it is known that a very low shear rate can enhance the formation of aggregates, as demonstrated by the modal relationship of the shear-rate dependence of RBC aggregation. The objective of the present study was, thus, to evaluate the influence of sudden flow reductions compared to a complete flow stop...
Vidugiris GJ, Royer CA.The volume change for the transition from the native state of horse heart apomyoglobin to a pressure-induced intermediate with fluorescence properties similar to those of the well-established molten globule or I form was measured to be -70 ml/mol. Complete unfolding of the protein by pressure at pH 4.2 revealed an upper limit for the unfolding of the intermediate of -61 ml/mol. At 0.3 M guanidine hydrochloride, the entire transition from native to molten globule to unfolded state was observed in the available pressure range below 2.5 kbar. The volume change for the N-->I transition is relat...
Ebert DW, Roberts C, Farrar SK, Johnston WM, Litsky AS, Bertone AL.Measurements of absolute total reflectance were recorded from weight-bearing (n=9) and nonweight-bearing (n=9) equine articular cartilage specimens from 300 to 850 nm using a spectrophotometer with integrating sphere attachment. Following correction of measured spectra for interfacial reflections and edge losses, Kubelka-Munk theory was applied to estimate absorption and scattering coefficient, one-dimensional light intensity distribution, and light penetration depth. Kubelka-Munk absorption coefficients ranged from ∼7 cm-1 at 330 nm to ∼1 cm-1 at 850 nm. A localized absorption peak wa...
Takai S, Vigo G, Ikushima H, Higuchi T, Hagiwara S, Hashikura S, Sasaki Y, Tsubaki S, Anzai T, Kamada M.Polymerase chain reaction (PCR)-based assays were developed to detect virulent Rhodococcus equi in transtracheal aspirate samples from sick foals showing respiratory signs. An oligonucleotide primer pair from the sequence of the virulence-associated 15- to 17-kDa antigen gene of the virulence plasmid in virulent R. equi was used to amplify a 564 bp region by PCR, and the result was confirmed by Southern blot hybridization. No positive reaction was seen in DNA from 13 different microorganisms typically found in the respiratory tract. In tracheal aspirates seeded with virulent R. equi, a visible...
Ilobi CP, Nicolson C, Taylor J, Mumford JA, Wood JM, Robertson JS.Equine influenza viruses propagated in the laboratory in alternate hosts such as embryonated hens' eggs or mammalian cell culture have been analysed by HA sequencing and antigenically and their sequence compared to the original virus present in clinical material. In contrast to clinically derived human influenza virus which generally grows in MDCK cells without change, the data for equine influenza virus were less clear in that variants of equine virus were derived in both eggs and cells. The study indicated that the current use of eggs for equine influenza virus surveillance and vaccine produ...
Bird J, Larsen M, Nansen P, Kraglund HO, Grønvold J, Henriksen SA, Wolstrup J.Two sets of dung-derived organisms from soil routinely fertilized with manure (MA) and soil chemically fertilized (CH) were cultured separately in the laboratory. Baermannized organisms from these cultures were added to 20 g of faeces from strongyle-infected horses to form three treatment groups: (i) no soil organisms; (ii) low inoculum of soil organisms containing all organisms present in a suspension of approximately 100 adult female free-living nematodes; and (iii) high inoculum containing those soil organisms present with approximately 1000 adult female free-living nematodes. Three studies...
Peauroi JR, Fisher DJ, Mohr FC, Vivrette SL.A 14-year-old Arabian gelding had weight loss and anorexia of 3 weeks' duration. Results of repeated laboratory tests revealed persistent hypercalcemia and serum phosphorus concentration that was within or less than the reference range. Parathyroid hormone concentration was high. Histologic examination of specimens obtained at necropsy revealed parathyroid adenoma. A diagnosis of primary hyperparathyroidism attributable to a functional parathyroid adenoma was made. Abnormalities in calcium and phosphorus concentrations were similar to those seen with primary hyperparathyroidism in dogs, in whi...
Pellegrini A, Kalkinc M, Hermann M, Grünig B, Winder C, Von Fellenberg R.Equinins are a closely related group of proteins found in equine neutrophil granules. They demonstrate proteinase inhibiting activity restricted to microbial proteinase K and subtilisin, and they also possess antibacterial and antiviral properties. Antiproteinase K activity was measured in tracheobronchial secretions (TBS) of horses with mild (n = 15), moderate (n = 30) and severe (n = 16) chronic pulmonary disease, to determine its usefulness as an indicator of severity of disease and to measure neutrophil content. Determination of proteinase K inhibiting activity was based on a colorimetric ...
Brightwell G, Brown JM, Coates DM.Rt-PCR probes targeted to different gene sequences of VEE (Venezuelan equine encephalitis) virus strain TC-83 were assessed for their sensitivity, specificity and non-specific cross-reactivity. A generic VEE virus amplimer (VNSP4F2/VNSP4R2), targeted against nsP4 was identified, which was sensitive (detected at least 10 pfu) and robust (worked over a wide range of salt concentrations and annealing temperatures). An E2 amplimer designed against TC-83, (VE2F/VE2R), identified VEE strains TRD (1AB), P676 (1C), 3880 (1D) Everglades (2) vRNA whilst a second E2 primer pair designed against strain 68...
Sheoran AS, Lunn DP, Holmes MA.This paper describes the production of a panel of monoclonal antibodies (mAbs) identifying the four recognised equine IgG subisotypes IgG, IgGa, IgGb, IgGc and IgG(T). Pure preparations of the subisotypes for use in immunisations and testing were produced using a combination of gel filtration, salt precipitation, ion exchange chromatography and protein A and Protein G affinity chromatography. The specificity of mAbs for the IgG subisotypes was confirmed using ELISA assays, by characterisation of affinity purified proteins recognised by the mAbs, and by Western blotting of equine serum proteins...
Bhuyan AK, Udgaonkar JB.The unfolding kinetics of horse cytochrome c in the oxidized state has been studied at 10, 22, and 34 degreesC as a function of guanidine hydrochloride (GdnHCl) concentration. Rapid (millisecond) measurements of far-UV circular dichroism (CD) as well as fluorescence quenching due to tryptophan to heme excitation energy transfer have been used to monitor the unfolding process. At 10 degreesC, the decrease in far-UV CD signal that accompanies unfolding occurs in two phases. The unobservable burst phase is complete within 4 ms, while the slower phase occurs over tens to hundreds of milliseconds. ...
Raabe MR, Issel CJ, Montelaro RC.Equine infectious anemia virus (EIAV) has been shown to infect cells of monocyte/macrophage lineage. These primary cells are intrinsically difficult to obtain, to purify and to culture in vitro for extended periods of time. As a result, most in vitro studies concerning this lentivirus make use of primary equine fibroblasts or transformed canine or feline cell lines. We describe methods that yield reproducibly pure cultures of equine blood monocytes from peripheral blood mononuclear cells. The in vitro differentiation of these cells into mature equine macrophage was verified using various cytoc...
Granier T, Comberton G, Gallois B, d'Estaintot BL, Dautant A, Crichton RR, Précigoux G.We refined the structure of the tetragonal form of recombinant horse L-chain apoferritin to 2.0 A and we compared it with that of the cubic form previously refined to the same resolution. The major differences between the two structures concern the cadmium ions bound to the residues E130 at the threefold axes of the molecule. Taking advantage of the significant anomalous signal (f" = 3.6 e-) of cadmium at 1.375 A, the wavelength used here, we performed anomalous Fourier difference maps with the refined model phases. These maps reveal the positions of anomalous scatterers at different locations...
Howard RD, McIlwraith CW, Trotter GW, Nyborg JK.To clone equine interleukin 1 receptor antagonist (IL-1ra) and determine its full-length cDNA sequence. Methods: A cDNA library derived from lipopolysaccharide-stimulated equine monocytes was screened by means of plaque hybridization to radiolabeled equine IL-1ra DNA probes generated by means of the polymerase chain reaction. The cDNA nucleotide sequence for equine IL-1ra was determined by use of the dideoxy chain termination technique, analyzed by use of computer software for sequence characteristics, and compared with sequences reported for IL-1ra of other species. Results: The cDNA of equin...
Valberg SJ, Townsend D, Mickelson JR.To determine whether polysaccharide storage myopathy (PSSM) in Quarter Horses is attributable to a defect in glycolysis or in the allosteric regulation of phosphofructokinase (PFK) enzyme. Methods: Muscle biopsy specimens were obtained from 6 Quarter Horses with PSSM and 8 Quarter Horse or Thoroughbred control horses. Methods: Maximal activity of glycogenolytic and glycolytic enzymes was determined spectrophotometrically. Maximal activity of PFK was determined for each horse at pH 8.0, and at pH 7.0 when variable concentrations of the activators, fructose 6 phosphate, fructose 2,6 bisphosphate...
Howard RD, McIlwraith CW, Trotter GW, Nyborg JK.To clone equine interleukin 1 alpha (IL-1 alpha) and equine interleukin 1 beta (IL-1 beta) and determine their full-length cDNA sequences. Methods: The mRNA isolated from lipopolysaccharide-stimulated cultured equine monocytes was reverse transcribed, and a cDNA library was constructed in a lambda phage. The cDNA library was screened by means of plaque hybridization with radiolabeled human IL-1 alpha and IL-1 beta cDNA probes. The cDNA nucleotide sequences for equine IL-1 alpha and equine IL-1 beta were determined by use of the dideoxy chain termination technique. The cDNA sequences were analy...
Brown MP, West LA, Merritt KA, Plaas AH.To determine effects of aging on sulfation of chondroitin sulfate (CS) in articular cartilage and synovial fluid from normal equine middle carpal joints, and to determine whether CS compositional analysis can be used to assess alterations in proteoglycan turnover in degenerative cartilage obtained from horses with carpal osteochondral fractures. Methods: Carpal articular cartilage and synovial fluid from 44 cadavers with normal joints and from 16 Thoroughbred racehorses during routine carpal arthroscopic surgery. Methods: After papain/chondroitinase digestion of cartilage, CS disaccharides (un...
Brink P, DeGraves F, Ravis WR, Johansen D, Campbell JD, Duran SH.To determine intravascular and intrasynovial pharmacokinetics of the R and S enantiomers of ketoprofen after i.v. and i.m. administration to horses. Methods: 6 healthy adult mares. Methods: Horses were weighed and ketoprofen (2.2 mg/kg of body weight) was administered i.v. Blood and synovial fluid samples were obtained and analyzed for concentrations of the R and S enantiomers by means of a modified reverse-phase stereospecific high-pressure liquid chromatographic method. Three weeks later, the procedure was repeated, except that ketoprofen was given IM. Protein binding of ketoprofen enantiome...
Reubel GH, Barlough JE, Madigan JE.We report on the production and characterization of Ehrlichia risticii, the agent of Potomac horse fever (PHF), from snails (Pleuroceridae: Juga spp.) maintained in aquarium culture and compare it genetically to equine strains. Snails were collected from stream waters on a pasture in Siskiyou County, Calif., where PHF is enzootic and were maintained for several weeks in freshwater aquaria in the laboratory. Upon exposure to temperatures above 22 degrees C the snails released trematode cercariae tentatively identified as virgulate cercariae. Fragments of three different genes (genes for 16S rRN...
Hatzipanagiotou A, Rieland E, Enbergs H.It was the aim of this project to investigate the changes of the lysozyme activity in the milk of mares during the lactation period. Further on the influence of race, date of conception and foaling, age and number of lactations on the lysozyme activities in milk was analysed. Milk samples were collected from 44 mares (trotters, warmblood, quarter horses) from eight farms between the 1st and 90th day p. p. The activity of the lysozyme was measured by a turbidometric method. Summarizing the following results are obtained: Lysozyme activities in mare milk of the 1st and 3rd day p. p. were higher ...
Henckel P, Ducro B, Oksbjerg N, Hassing L.The objectivity of two of the most widely used methods for differentiation of fibre types, i.e. 1) the myosin ATP-ase method (Brooke and Kaiser, 1970a,b) and 2) the combined method, by which the myosin ATP-ase reaction is used to differentiate between fast and slow twitch fibres and NADH-tetrazolium reductase activity is used to identify the subgroups of fast twitch fibres (Ashmore and Doerr, 1970, Peter et al., 1972), was assessed in muscle samples from horses, calves and pigs. We also assessed the objectivity of the alpha-amylase-PAS preparation for the visualisation of capillaries (Andersen...
Yachida Y, Kashiwagi M, Mikami T, Tsuchihashi K, Daino T, Akino T, Gasa S.Modified galactosylceramide with a long-chain cyclic acetal at the sugar moiety, plasmalogalactosylceramide, was isolated from equine brain. To identify the isomeric stereostructure of the natural product, the plasmalo derivative was chemically synthesized from galactosylceramide through acetalization. The presence of cyclic acetal linkage, the linked position and length of the acetal chain of the synthesized and natural products were determined by proton nuclear magnetic resonance spectroscopy and fast-atom bombardment-mass spectrometry, as well as gas chromatography-mass spectrometry and gas...
Flanagan J, Collin N, Timoney J, Mitchell T, Mumford JA, Chanter N.The haemolytic activity of Streptococcus equi, the cause of equine strangles, was characterized. Production of haemolysin in Todd Hewitt broth was dependent on an equine serum supplement and the logarithmic phase of growth after which activity declined sharply. RNA core also induced haemolysin production from cells harvested at the end of the logarithmic phase of growth. Haemolysis was not affected by cholesterol, was only slightly increased in reducing conditions and was completely inactivated by trypan blue, identifying the haemolytic activity as streptolysin S-like (SLS-like). Purification ...
Otte K, Choudhury D, Charalambous M, Engström W, Rozell B.The equine IGF2 gene has been cloned and characterised. It spans a 9 kb region, which is substantially less than the corresponding human gene. Three coding exons and three untranslated leader exons, all highly homologous to those in other species, were identified. Downstream of the polyadenylation site in exon 6, a dinucleotide repeat sequence was identified. Three putative promoters (P1-P3) were localised in the 5' region of the gene. RNase protection analysis revealed two active promoters in fetal tissues, P2 and P3, whereas P3 was the only promoter active in adult tissues. This represents a...
Willbold D, Metzger AU, Sticht H, Gallert KC, Voit R, Dank N, Bayer P, Krauss G, Goody RS, Rösch P.Lentiviral transactivator (Tat) proteins are essential for viral replication. Tat proteins of human immunodeficiency virus type 1 and bovine immunodeficiency virus form complexes with their respective RNA targets (Tat responsive element, TAR), and specific binding of the equine anemia virus (EIAV) Tat protein to a target TAR RNA is suggested by mutational analysis of the TAR RNA. Structural data on equine infectious anemia virus Tat protein reveal a helix-loop-helix-turn-helix limit structure very similar to homeobox domains that are known to bind specifically to DNA. Here we report results of...
Mikami T, Kashiwagi M, Tsuchihashi K, Daino T, Akino T, Gasa S.Equine brain gangliosides were isolated and their structures were characterized, to examine whether equine brain has N-glycolyl neuraminic acid in gangliosides, since other mammals predominantly possess N-acetyl neuraminic acid in brain gangliosides, and equine erythrocytes and organs except the brain have gangliosides exclusively containing N-glycolyl neuraminic acid. The gangliosides purified from the brain were identified by proton NMR spectroscopy and mass spectrometry, as well as GLC, resulting in their identification as GM4, GM3, GM2, GM1, GD1a, GD1b, and GT1b. Of these gangliosides, GM3...
Hinrichs K.When recovered from the follicle, horse oocytes may be categorised as having either a compact or an expanded cumulus. Cumulus expansion is strongly associated with follicle atresia. Oocytes with expanded and compact cumuli have similar proportions in the germinal vesicle stage when recovered from the follicle. However, during in vitro culture, a higher proportion of oocytes with expanded cumuli mature, and they do so more quickly, than do oocytes with compact cumuli. Using Hoechst 33258 to label chromatin, in the germinal-vesicle stage horse oocytes can be divided into those in which the nucle...
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...
Aguilar JJ, Woods GL, Miragaya MH, Olsen LM.The object of this experiment was to estimate the number and type of living cells in oviductal masses of mares. Oviducts of abattoir mares were dissected, divided into 3 sections, and flushed individually. Oviductal masses were recovered from 220 of 250 mares and from 389 of 500 oviducts. A greater number of masses was recovered from the left than the right oviducts. A higher percentage of masses was recovered from the ampullary-isthmic junction than from the ampulla or isthmus. The number of masses increased slightly with increasing mare age and was weakly correlated with the number of unfert...
Brinkmann J, Koudelka T, Keppler JK, Tholey A, Schwarz K, Thaller G, Tetens J.The production and consumption of mare's milk in Europe has gained importance, mainly based on positive health effects and a lower allergenic potential as compared to cows' milk. The allergenicity of milk is to a certain extent affected by different genetic variants. In classical dairy species, much research has been conducted into the genetic variability of milk proteins, but the knowledge in horses is scarce. Here, we characterize two major forms of equine αS2-casein arising from genomic 1.3 kb in-frame deletion involving two coding exons, one of which represents an equid specific duplicati...
Ramsay JD, Leib SR, Orfe L, Call DR, Tallmadge RL, Fraser DG, Mealey RH.Development of an accurate and efficient molecular-based equine MHC class I typing method would facilitate the study of T lymphocyte immune responses in horses. Here, a DNA microarray was designed to detect expressed classical MHC class I genes comprising serologically defined equine leukocyte antigen (ELA)-A haplotypes represented in a closed Arabian horse breeding herd. Initially, cloning and sequencing of RT-PCR products were used to identify sequences associated with the ELA-A1, A4, and W11 haplotypes, and one undefined haplotype, in six horses. Subsequently, sequence-specific, conserved (...
Bertelsmann H, Sieme H, Behne D, Kyriakopoulos A.In the sperm nuclei, of mammalian species selenium has been found only in the form of sperm nuclei glutathione peroxidase (snGPx) where it is most likely bound to the chromatin of spermatozoa. Over 80% of selenium in sperm is bound to the selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) in the midpiece of rat sperm. Zinc in sperm is mainly contained in the outer dense fiber (ODF) proteins of the flagella of mammalian spermatozoa. In the sperm nuclei, zinc is predominately located in the chromatin to the protamine proteins. In order to investigate if the insertion of zinc...
Ferrante M, Andreeta A, Landoni MF.Diclofenac is a hydrophilic non-steroidal anti-inflammatory drug widely used in humans and animals. Previous reports have shown that this compound has low percutaneous absorption in horses. The effect of five penetration enhancers (10% urea, 15% and 20% oleic acid and 5% and 10% d-limonene) on the percutaneous absorption of diclofenac diethylamine through horse skin was evaluated in vitro using Franz-type diffusion cells. All tested penetration enhancers induced a significant increase in diclofenac diethylamine permeation, with limonene showing the highest enhancing effect at the lowest concen...
Miteva MA, Kossekova GP, Villoutreix BO, Atanasov BP.The present work shows the application of an optical label pyridoxal phosphate (PLP) for the experimental determination of local electrostatic potentials in singly substituted cytochromes c modified by pyridoxal phosphate at Lys 79 (PLP-Lys-79-cyt.c) or at Lys 86 (PLP-Lys-86-cyt.c). PLP has also been used to calculate the pKa values of all ionizable groups and the electrostatic potentials in the modified proteins and to analyse their properties. The experimental pKa values for the pyridine nitrogen and phenolic hydroxyl of the bound label were obtained from pH-dependent absorbance and fluoresc...
Hans A, Gaudaire D, Manuguerra JC, Leon A, Gessain A, Laugier C, Berthet N, Zientara S.This study shows that an unbiased amplification method applied to equine arteritis virus RNA significantly improves the sensitivity of the real-time reverse transcription-quantitative PCR (RT-qPCR) recommended by the World Organization for Animal Health. Twelve viral RNAs amplified using this method were hybridized on a high-density resequencing microarray for effective viral characterization.
Okamura M, Yokoyama N, Takabatake N, Okubo K, Ikehara Y, Igarashi I.In the present study, we investigated the effects of protease pretreatments of host erythrocytes (RBC) on the in vitro growth of bovine Babesia parasites (Babesia bovis and B. bigemina) and equine Babesia parasites (B. equi and B. caballi). The selected proteases, trypsin and chymotrypsin, clearly modified several membrane proteins of both bovine and equine RBC, as demonstrated by SDS-PAGE analysis; however, the protease treatments also modified the sialic acid content exclusively in bovine RBC, as demonstrated by lectin blot analysis. An in vitro growth assay using the protease-treated RBC sh...
Herrera C, Morikawa MI, Castex CB, Pinto MR, Ortega N, Fanti T, Garaguso R, Franco MJ, Castañares M, Castañeira C, Losinno L, Miragaya MH, Mutto AA.Normal mammalian early embryonic development involves apoptosis of blastomeres as a remodeling process during differentiation, starting at the blastocyst stage. Genomic DNA has been recently detected in the blastocele fluid of human embryos and has been amplified by real-time polymerase chain reaction (PCR) to diagnose the sex of in vitro-produced human embryos. This new approach varies from conventional preimplantation genetic diagnosis in that no cells are extracted from the embryo and only the blastocele fluid is aspirated and used as a DNA sample for diagnosis. In the present work, we inv...
Clegg PD, Jones MD, Carter SD.Loss of articular cartilage, which is the most important pathological lesion occurring in osteoarthritis, has been shown to be enzymatically mediated. The matrix metalloproteinases (MMPs) are a group of enzymes which have been implicated in this degradation of articular cartilage matrix. The use of pharmacological agents to inhibit this catabolic process in the joint is a potential route for therapeutic intervention. The gelatinase MMPs, MMPs-2 and 9, were purified by affinity chromatography from equine cell cultures. The ability of phenylbutazone, flunixin, betamethasone, dexamethasone, methy...
Peters GJ, De Abreu RA, Oosterhof A, Veerkamp JH.Concentrations of purine and pyrimidine ribonucleotides were measured with HPLC in lymphocytes of man, horse, pig and sheep and in rat thymocytes. The ATP concentration was highest in lymphocytes of all species and about 850 pmol/10(6) cells in human and equine lymphocytes, higher in porcine and lower in ovine lymphocytes and rat thymocytes. The GTP concentration was comparable in human, equine and porcine lymphocytes, but lower in ovine lymphocytes. ATP concentration was also measured in lymphocytes of man, horse and pig with a luciferin-luciferase assay. During culturing with or without phyt...
Lombardo D, Chapus C, Bourne Y, Cambillau C.Horse (Equus caballus) pancreatic lipase (EC 3.1.1.3) has been crystallized using the hanging drop method of vapour diffusion at 20 degrees C. The best crystals were grown from an 8 mg/ml solution in 10 to 20% (w/v) polyethylene glycol 8000, 10 mM-MgCl2, 0.1 M-NaCl, 0.1 M-Mes buffer (pH 5.6). They reach dimensions of 0.8 mm x 0.4 mm x 0.6 mm. X-ray examination of the lipase crystals shows that they are orthorombic with a space group P2(1)2(1)2(1). Their cell dimensions are a = 79.8 A, b = 97.2 A c = 145.3 A. Two molecules per asymmetric unit give a Vm value of 2.82 A3/dalton (56% water content...
Castro T, Jacob JC, Domingues RR, Ginther OJ.Prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) are involved in equine embryo mobility throughout the uterus on Days 11-15 (ovulation = Day 0). On a day (Day 12) of maximal embryo mobility in pregnant mares (n = 13) and before luteolysis in nonbred mares (n = 10), gene expressions were compared between the uterine horns that did and did not contain the mobile embryo and between pregnant and nonbred mares. A cytobrush was used to collect an endometrial sample from the middle of each uterine horn. In nonbred mares, there was no difference for any of the considered gene expressions ...
Sugiura T, Sugita S, Imagawa H, Kanaya T, Ishiyama S, Saeki N, Uchiyama A, Tanigawa M, Kuwano A.The hemagglutinin (HA) protein of an equine influenza strain, A/equine/La Plata/1/93 (LP/93), was produced using a baculovirus expression system. Silkworm larvae inoculated with recombinant baculovirus expressed high quantities of the HA protein which was then purified to greater than 95% purity by fetuin-affinity chromatography. Purified HA protein was used subsequently in an ELISA for detection of antibodies in horse sera. Two hundred serum samples from vaccinated racehorses were reacted on ELISA plates coated with 40.0 ng/ml of purified HA protein. Subsequent optical density (OD) levels rev...
Johannisson A, Gröndahl G, Demmers S, Jensen-Waern M.Methodological aspects of flow-cytometric evaluation of the phagocytic properties of equine neutrophils were elucidated. The kinetics of attachment and ingestion were studied, and the phagocytic process was more rapidly completed when serum-opsonized yeast cells were used than with use of IgG-opsonized yeast cells. Trypan blue was successfully used to quench fluorescence of non-ingested yeast cells. There were only minor differences in the kinetics of phagocytosis between quenched and unquenched samples, indicating that attachment is rapidly followed by ingestion. Trypan blue quenching caused ...
Epstein KL, Brainard BM.Accurate measurement of plasma fibrinogen concentrations is an important tool for assessment of horses with inflammatory diseases. Objective: To determine the precision and accuracy of a benchtop instrument using both fresh and frozen equine plasma by comparing the plasma fibrinogen concentration measured by a benchtop instrument to 2 separate laboratory standard methods (ACL 100 and STA Compact) for fibrinogen measurement. Methods: Accuracy and precision of the VSPro was evaluated using both human fibrinogen standards and samples from horses. Fifty frozen samples from horses with gastrointest...
Cerón JJ, Tecles F, Espín JC.Effects of seven different blood diluents (distilled water, Triton X-100, saponin, isotonic saline solution, pH 7.5 and 8 phosphate buffers and bovine serum albumin) and two chromophores: 5, 5'-dithiobis 2-nitrobenzoic acid (DTNB) and 2,2'-dithiodipyridine (2- PDS) on blood cholinesterase determination in four domestic species (cow, sheep, goat and horse) are described and compared. Haemolytic diluents (distilled water, Triton X-100 and saponin) gave the best precision results when fresh blood was assayed. However, Triton X-100 induced lower ChE activity values in horses, and saponin yielded v...
Jones I, Madej A.Microtitre plates were coated with antiserum against oestradiol-17 beta-6-(O-carboxymethyl)-oxime bovine serum albumin raised in sheep. The plasma samples (0.2-1.0 ml) were extracted with peroxide-free diethyl ether prepared daily by treatment with Al2O3. The enzyme conjugate was prepared by coupling oestradiol-17 beta-6-(O-carboxymethyl)-oxime to horse-radish peroxidase. The conjugate was chromatographed on a Sephadex G-25 column. The standard curve ranged from 0.37 to 18.40 fmol/well of oestradiol-17 beta. The amount of oestradiol-17 beta causing a 50% reduction of maximum binding was 4.4 fm...
Hernández-Vidal G, Jeffcott LB, Davies ME.A polyclonal antiserum raised in sheep against human cathepsin B was tested for specificity and cross-reactivity with the horse homologue by SDS-PAGE and Western blotting, prior to being used for immunolocalization of the enzyme in equine articular cartilage. In Western blots, the antiserum recognized the 30 kDa single chain and 25 kDa heavy chain of the mature enzyme in purified bovine cathepsin B, and corresponding bands at 32 and 27 kDa in equine chondrocyte and fibroblast lysates. This antiserum was then used to compare the expression and distribution of cathepsin B in normal and dyschondr...
Fontes KFLP, Silva-Júnior LC, Nascimento SA, Chaves DP, Pinheiro-Júnior JW, Freitas AC, Castro RS, Jesus ALS.A codon-optimized equine infectious anemia virus p26 gene was fused to a maltose-binding protein (MBP) and expressed in Escherichia coli for use as an antigen in agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) for diagnosis of equine infectious anemia. An analysis of analytical sensitivity and specificity showed that the antigen MBP-p26rec reacted positively with a reference World Organization for Animal Health serum and demonstrated no cross-reaction against sera from vaccinated animals in either test. The diagnostic characteristics were evaluated and presented e...
Bousfield GR, Liu WK, Ward DN.LH hybrids were prepared by combining eLH alpha and eLH beta with the corresponding subunits of oLH, pLH and hCG. Recombinants were isolated by gel filtration and assessed by SDS-polyacrylamide gel electrophoresis under both dissociating and non-dissociating conditions. All combinations of subunits produced hybrid LH molecules. Hybrids prepared by combining eLH beta with oLH alpha, pLH alpha or hCG alpha were very inactive in rat radioligand and Leydig cell in vitro bioassays. Hybrids prepared with eLH alpha were very active in both assays. The greatest potentiating activity was observed when ...
Moiseeva SA, Postnikova GB.The influence of Cu2+ concentration, pH, and ionic strength of the solution as well as redox-inactive zinc ions on the rate of oxidation of sperm whale, horse, and pig oxymyoglobins (oxy-Mb) by copper ions has been studied. These myoglobins have homologous spatial structures and equal redox potentials but differ in the number of histidines located on the surface of the proteins. It was shown that oxy-Mb can be oxidized in the presence of Cu2+ through two distinct pathways depending on which histidine binds the reagent and how stable the complex is. A slow pH-dependent catalytic process is obse...
Richard F, Martinat N, Remy JJ, Salesse R, Combarnous Y.Among all mammalian FSH receptors (FSH-R; including donkey (dk) FSH-R), only horse (hs) FSH-R does not bind hsLH/chorionic gonadotrophin (CG). In order to delineate the structural origin of hsFSH-R specificity precisely, we have cloned dkFSH-R cDNA from donkey testis mRNA by RT-PCR. Transiently expressed dkFSH-R endowed COS-7 cells with both hsLH/CG- and FSH-binding activity, as well as FSH-induced cAMP production. The deduced dkFSH-R amino acid sequence shares 96% identity with the hsFSH-R: notably, in the hormone-binding domain, the specificity of hsFSH-R may be ascribed to only four diverge...
Parrilla Hernández S, Franck T, Munaut C, Feyereisen É, Piret J, Farnir F, Reigner F, Barrière P, Deleuze S.Myeloperoxidase (MPO), as a marker of neutrophil activation, has been associated with equine endometritis. However, in absence of inflammation, MPO is constantly detected in the uterine lumen of estrous mares. The aim of this study was to characterize MPO in the uterus of mares under physiological conditions as a first step to better understand the role of this enzyme in equine reproduction. Total and active MPO concentrations were determined, by ELISA and SIEFED assay, respectively, in low-volume lavages from mares in estrus ( = 26), diestrus ( = 18) and anestrus ( = 8) in absence of endometr...
Butler WF, Pousty I.Disc material from horse, ox, sheep, pig, dog and cat was stained by the Alcian-blue-critical electrolyte concentration technique and with the standard and two-step periodic acid Schiff methods. The effects of pretreatment with hyaluronidase and with chondroitinase was also evaluated. There appears to be a small increase in total cellular glycosaminoglycan content with age in all species: cellular material of high molecular weight however only increases in aged animals. The degree of sulphation of cellular glycosaminoglycans does not vary with age or with position in the disc.
Sedgwick AD, Lees P, Dawson J, May SA.The migration of leucocytes to sites of acute and chronic inflammation is an event of central importance to the maintenance of inflammatory processes; extravascular leucocytes are responsible for generating chemical mediators of inflammation and the phagocytosis of particulate matter. They may also be involved in the conversion of acute to chronic inflammatory lesions. Leucocytes are attracted to sites of tissue injury by a range of chemoattractants. This paper describes the development of a method for separating on Percoll gradients purified populations of equine polymorphonuclear and mononuc...
Theurillat R, Sandbaumhüter FA, Gittel C, Larenza Menzies MP, Braun C, Thormann W.An enantioselective assay for the determination of methadone and its main metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in equine plasma based on capillary electrophoresis with highly sulfated γ-cyclodextrin as chiral selector and electrokinetic analyte injection is described. The assay is based on liquid/liquid extraction of the analytes at alkaline pH from 0.1 mL plasma followed by electrokinetic sample injection of the analytes from the extract across a buffer plug without chiral selector. Separation occurs cationically at normal polarity in a pH 3 phosphate buffer containin...
Brooks BW, Lutze-Wallace CL, Maclean LL, Vinogradov E, Perry MB.Lipopolysaccharides (LPSs) from Taylorella equigenitalis, the causative agent of contagious equine metritis, and T. asinigenitalis were compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Lipopolysaccharide profiles of 11 T. equigenitalis strains were similar, but different from the profiles of 3 T. asinigenitalis strains, and the profiles of 2 T. asinigenitalis strains were similar to each other. The serological specificities of the LPSs from these 14 strains were examined by immunoblotting and enzyme-linked immunosorbent assay with monoclonal antibodies (MAbs) ...
Charbord P, Tippens D, Wight TS, Gown AM, Singer JW.This report describes an IgG1 mouse monoclonal antibody derived after immunization of mice with washed stromal cells from human, long-term bone marrow cultures. The antigen recognized by the antibody (BMS-1) is a carbohydrate-containing prosthetic group that is common to and specific for multiple horse serum proteins. These proteins are avidly ingested by stromal cells and concentrated in endocytic vesicles. Cultured smooth muscle cells took up the horse proteins in a similar manner to marrow stromal cells while cultured marrow fibroblasts, endothelial cells, and hepatoma cells did not. These ...
Ishiyama T, Shinagawa M, Sato G, Fujinaga K, Padmanabhan R.Equine adenovirus (EAd) DNA prepared from infected bovine kidney (MDBK) cells contained additional sequences of about 100 to 700 bp at the left-hand end of the genome. These aberrant viral genomes were produced even after the first passage of the wild type EAd in MDBK cells and their relative amounts did not change significantly during serial passage. The left terminal fragments of two defective viral DNAs were cloned into the plasmid vector pBR322 and the nucleotide sequences of their terminal regions were analyzed. The data indicate that one viral DNA contained a duplication of the inverted ...
Chapus C, Desnuelle P, Foglizzo E.Pig and horse colipases have been purified by a common procedure using trypsin and carboxypeptidase inhibitors as stabilizers. Two forms of pig colipase were identified: a predominant A1 form with about 103-105 residues, and a minor slightly degraded A2 form in which the last two C-terminal residues, Asp and Ser, were lacking. This type of degradation is considerably slowed down by carboxypeptidase inhibitors. A total of four forms of the horse cofactor were characterized: two (A1 and B1) were probably isocolipases which differed by only a few substitutions. Both contained the same number of r...
