In vitro research involving horses refers to the study of equine cells, tissues, or biological molecules outside their normal biological context, typically in controlled laboratory environments. This research approach allows scientists to investigate cellular processes, molecular interactions, and the effects of various treatments without the ethical and logistical complexities of in vivo studies. In vitro studies contribute to understanding equine physiology, pathology, and pharmacology by providing insights into cellular responses to pathogens, drugs, and other stimuli. This page compiles peer-reviewed research studies and scholarly articles that explore various in vitro methodologies and their applications in equine science, including cell culture techniques, molecular assays, and drug efficacy testing.
Toniolo C, Fontana A, Scoffone E.Ultraviolet absorption and circular dichroism studies have been carried out on horse heart apo-cytochrome c and heme-free peptide fragments obtained by cyanogen bromide cleavage of the native protein. It was noted that the various peptides assume predominantly an unordered conformation in water solution. Increasing ionic strength and addition of 2-chloroethanol increase the right-handed helical content. Guanidinium hydrochloride favors the coil state. It was also demonstrated that two non-interacting helical regions of different stability are present in the apo-protein in 2-chloroethanol.
Levitt NH, Miller HV, Pedersen CE, Eddy GA.The development of a new diagnostic procedure for the identification of Venezvelan, eastern and western equine encephalomyelitis (VEE, EEE, WEE) viruses is described. The procedure utilizes virus precipitation with reference fluorescein-conjugated gamma globulin, followed by cellulose acetate electrophoresis. Clinical specimens containing varying concentrations of virus yielded, in primary duck embryo cell culture, sufficient virus for detection within 22 to 44 hours. Identification of VEE, EEE and WEE virus in specimens was accomplished by microprecipitation within this time. In contrast to c...
Péterfy F, Varró R, Fatrai Z, Barna I, Kiss I.Horse immune sera do not give satisfactory results in immunochemical techniques based on electrophoresis of antigens through antibody-containing agarose gel. As the majority of precipitating horse antibodies belongs to the beta globulins, they migrate in the gel during electrophoresis. After enzymatic treatment the pepsin fragments work well in all electroimmunodiffusion methods.
Boorman J, Mellor PS, Penn M, Jennings M.Seven-day-old embryonated hen eggs were infected with African Horse Sickness virus by the yolk sac and intravenous routes. Virus reached a high titre in the blood of infected embryos. Culicoides variipennis midges which took a blood meal from infected eggs became infected with virus, and after 7 days at 26 degrees - 27 degrees C transmitted African Horse Sickness virus to uninfected eggs. C. variipennis may therefore be considered a biological vector of African Horse Sickness virus in the laboratory.
Milgrom F, Loza U, Kano K.Double diffusion tests in gel were employed for studies of reactions between infectious mononucleosis sera and extracts of bovine, sheep and equine erythrocyte stromata. The extracts were obtained by ultrasonication of stromata prepared from trypsin-digested erythrocytes. The reaction with bovine stroma extract was composed, in many instances, of two lines. A single line was observed in reactions with sheep and equine stroma extracts. This line merged into a reaction of partial or complete identy with one of the lines formed with bovine stroma extract. Evidence was obtained that some infectioo...
Oeding P, Hájek V, Marsálek E.Out of 70 S. aurew strains isolated from the anterior nares of horses, 48 (69 per cent)
belonged to the E biotype. Approximately one third of these isolates were typed with factor
sera, the 6 (35 per cent) that were typable showing 5 different patterns. All strains but one
were non-typable with the basic sets of phages for typing human and bovine staphylococci
even at RTD x 100. Without any exception the equine staphylococci of the E biotype
contained polysaccharide Aa. Sixteen biochemically different strains belonged to the biotype A, B or C. A number of different serological patterns an...
Augustinsson KB, Bartfai T, Mannervik B.The steady-state kinetics of the butyrylcholinesterase-catalysed hydrolysis of butyrylthiocholine and thiophenyl acetate were shown to deviate from Michaelis-Menten kinetics. The ;best' empirical rate law was selected by fitting different rate equations to the experimental data by non-linear regression methods. The results were analysed in view of two alternative interpretations: (1) the reaction is catalysed by a mixture of enzymes, or (2) the activity is due to a single enzyme displaying deviations from Michaelis-Menten kinetics. It was concluded that the second alternative applies, and this...
Margalit R, Schejter A.The specific binding of cations to horse heart ferrocytochrome c has been studied, using the gel
filtration method. The cations investigated were: Mg2+, Co2+, cinchonine and proflavine. The stability
constants are in the range of 5-8 mM-1, and the number of binding sites per protein molecule are
1 to 2. The temperature dependence of the stability constant for the Mg2+-ferrocytochrome system
was measured. The thermodynamic parameters were found to be: dH&s = 4-12 kcal/mol, dG;,,
(25 "C) = -5.6 kcal/mol and AS&, = +57 calxmol-lx K-I.
Wilgus H, Stellwagen E.Changes in the visible absorbance spectra of complexes of horse heart cytochrome c hemopeptide 1-65, peptide 66-104, and their guanidinated counterparts are compared with those characteristic of native and fully guanidinated ferricytochrome c over the pH range 7 to 11. Upon raising the pH, the methionine ligand in the guanidinated hemopeptide 1-65.peptide 66-104 complex is replaced by a strong field ligand. By contrast, the methionine ligand in the hemopeptide 1-65.guanidinated peptide 66-104 is replaced by a weak field ligand. These results demonstrate that lysine 13 does not ligate with the ...
Watson RE, Larson KA.Indirect immunofluorescence and lymphocyte cytotoxicity experiments demonstrated the presence of a tumor-specific antigen(s) on the surface of cells from an equine sarcoid cell line (Mc1). Autologous serum (taken from the horse from which the Mc1 cells were derived) and sera from three other sarcoid-bearing horses revealed a similar membrane immunofluorescence when reacted with Mc1 cells, indicating the existence of cross-reacting antibodies. Results of serum colony inhibition experiments indicate that these antibodies are not cytotoxic. Incubation of Mc1 cells with autologous lymphocytes resu...
England JJ, Watson RE, Larson KA.On electron microscopic examination of a cell line derived from an equine sarcoid, intracytoplasmic oncornavirus-like particles were seen. Cells treated with idoxuridine-dimethyl sulfoxide (idu-dmso) had a two- to four-fold increase in the number of particles as compared with nontreated cells or cells treated with dmso alone. The intracytoplasmic virus-like particles were double membrane structures measuring 80 to 100 nm. in diameter. Particles were seen extracelluarly or budding from the cell membrane into the extracellular space. These extracellular particles were 100 nm. in diameter and con...
Posthuma CC, Pedersen KW, Lu Z, Joosten RG, Roos N, Zevenhoven-Dobbe JC, Snijder EJ.The replication/transcription complex of the arterivirus equine arteritis virus (EAV) is associated with paired membranes and/or double-membrane vesicles (DMVs) that are thought to originate from the endoplasmic reticulum. Previously, coexpression of two putative transmembrane nonstructural proteins (nsp2 and nsp3) was found to suffice to induce these remarkable membrane structures, which are typical of arterivirus infection. Here, site-directed mutagenesis was used to investigate the role of nsp3 in more detail. Liberation of the hydrophobic N terminus of nsp3, which is normally achieved by c...
Kelling CL, Schipper IA, Schermeister LJ, Vacik JP.Extracts of 28 plants were tested without demonstable antiviral activity in an agar-overlay plaque-reduction antiviral assay system, using infectious bovine rhinotracheitis virus and bovine endocardial cell cultures. Ethanolic extract of Narcissus tazetta L bulb elicited antiviral activity by inhibition of viral plaque formation. Antiviral activity was demonstrated against infectious bovine rhinotracheitis and equine rhinopneumonitis viruses. Narcissus tazetta L bulb did not directly inactivate the virus extracellularly. The extract exhibited only limited toxicity to rapidly multiplying bovine...
Bentinck-Smith J, Tasker JB.The following topics are discussed in this presentation: A. Recent advances in the use and interpretation and methodology of antibiotic susceptibility testing. B. Improvements in sample submittal to obtain accurate results from your laboratory. C. Staining blood, bone marrow, and cytology specimens in the office laboratory. D. Pathogenesis and differential diagnosis of lipemia. E. Differential diagnosis of abdominal effusions.
Cunningham FM, Andrews M, Landoni MF, Lees P.Calcium ionophore A23187 induced time and concentration dependent production of immunoreactive leukotriene (LT) B4 by equine heparinized whole blood in vitro. Time dependent production of immunoreactive LTB4 by equine neutrophils and immunoreactive LTC4 by equine eosinophils in vitro was also demonstrated. The 5-lipoxygenase activating protein (FLAP) inhibitors, BAY X 1005 and BAY Y 1015, produced concentration dependent inhibition of ionophore-induced LTB4 synthesis by equine whole blood (mean +/- SEM IC50s n = 5; 6.14 +/- 0.28 microM vs. 12.30 +/- 0.75 microM for BAY Y 1015 and BAY X 1005, r...
Kearns CF, Lenhart JA, McKeever KH.Erythropoietin (EPO) is the primary hormone of erythropoiesis. Administration of recombinant human erythropoietin (rhuEPO) to improve racing performance in the horse represents a new form of blood doping, which has been associated with increased mortality. While immunoassay kits have become plentiful, very few commercial hormone assays are made specifically for equine research. There is a strong degree of sequence homology reported for EPO among species, which has allowed antibodies designed for human EPO research to be used to determine EPO concentration in other species. The objective of the...
Fukunaga Y, Wada R, Sugita S, Fujita Y, Nambo Y, Imagawa H, Kanemaru T, Kamada M, Komatsu N, Akashi H.Equine arteritis virus (EAV) was readily isolated in RK-13 cell monolayers by plaque assay from seminal plasma of experimental carrier stallions when they contained high titers of virus regardless of the presence of non-viral cytotoxicity in the seminal plasma. The cytotoxicity interfered with virus isolation from seminal plasma which contained virus at titers less than 10 PFU/ml. However, it was possible to detect the virus in seminal plasma pretreated with PEG (#6000). EAV was consistently identified by RT-PCR from crude seminal plasma which contained virus at titers of more than 10(2.7) PFU...
Jacobi S, Townsend WM, Bolin CA.To evaluate whether equine serum administered via a simulated subpalpebral lavage system (SPL) supports proliferation of Streptococcus zooepidemicus or Pseudomonas aeruginosa within the tubing. Methods: A sterile i.v. catheter with injection cap was inserted into sterilized silicone tubing (Mila). To mimic an SPL within the dorsal conjunctival fornix, the tubing was secured to an elevated platform. The tip of the tubing extended from the platform into a vial containing culture medium just inoculated with approximately 1.5 x 10(8) CFU/mL P. aeruginosa or S. zooepidemicus. To mimic administratio...
Söylemez Z, Ozer I.The inhibition of horse plasma cholinesterase by propranolol showed characteristics which depended upon the identity of the substrate used. With butyrylthiocholine as substrate, the inhibition showed a first-order dependence on inhibitor concentration, and was characterized by a Ki of 8 microM (pH 7.4, 20 degrees C). With p-nitrophenylbutyrate as substrate, a biphasic v-1 versus [I] relationship was obtained. The biphasic curve could be resolved into two components, with apparent Ki's of 9 microM and 1.3 mM. Use of butyrylthiocholine as alternative substrate resulted in partial inhibition of p...
Hayami A, Darwish WS, Ikenaka Y, Nakayama SM, Ishizuka M.There is little information about drug metabolism and pharmacokinetics in horses. Therefore, it is necessary to characterize the profiles of drug metabolites for the safe use of drugs. In this study, we focused on cytochrome P450 enzymes (CYPs), which represent an important enzyme group to determine pharmacological effects of drugs. We chose diazepam as the drug of choice for this study. The aim of this study was to elucidate the metabolic pathway of diazepam in horses in comparison with rats, and to clarify CYP subfamilies responsible for diazepam metabolism in horses. Our results showed tema...
Arns MJ, Webb GW, Kreider JL, Potter GD, Evans JW.Bovine serum albumin (BSA) diluents containing lactose, raffinose or sucrose were not different (P greater than 0.05) in their ability to maintain stallion sperm viability, as determined by percentage motile spermatozoa (PMS) and their rate of forward movement (RFM), when stored at 37 or 5 degrees C for 24 h. These diluents did promote a higher (P greater than 0.05) PMS and RFM, when compared with BSA diluents containing arabinose or galactose. The BSA-arabinose and BSA-galactose diluents did not differ (P less than 0.05) in their ability to support sperm viability and were detrimental to sper...
Schwab UE, Tallmadge RL, Matychak MB, Felippe MJB.OBJECTIVE To develop an in vitro system for differentiation of equine B cells from bone marrow hematopoietic progenitor cells on the basis of protocols for other species. SAMPLE Bone marrow aspirates aseptically obtained from 12 research horses. PROCEDURES Equine bone marrow CD34 cells were sorted by use of magnetic beads and cultured in medium supplemented with cytokines (recombinant human interleukin-7, equine interleukin-7, stem cell factor, and Fms-like tyrosine kinase-3), murine OP9 stromal cell preconditioned medium, and equine fetal bone marrow mesenchymal stromal cell preconditioned me...
Dimsoski P.To show that application of the polymerase chain reaction (PCR) method modified for amplification of a low-copy number DNA samples, ie, the isolation of PCR products (IPCRp), would represent improvement in obtaining genotypes from a fecal DNA compared with previously used genotyping methods. Methods: The DNA from the horse fecal matter was extracted by modified Qiagen DNA Stool Mini Kit protocol. Following the extraction, the DNA genotypes from fecal samples were obtained by the most powerful PCR amplification method, the IPCRp. The IPCRp-based multiplex kit amplified biotin-labeled strands we...
Sticht H, Willbold D, Rösch P.Two molecular dynamics (MD) simulations were performed in order to increase the understanding of the dependence of protein conformation on solvent environment. The protein used for these simulations is the transcriptional activator of the equine infectious anemia virus (EIAV-Tat). The structure of this protein has been determined by nuclear magnetic resonance (NMR) in aqueous solution (Willbold et al., Science 264, 1584 (1994)) and in 40% (v/v) trifluoroethanol (TFE) (Sticht et al., Eur. J. Biochem., submitted) showing considerable differences in the stability of the secondary structure elemen...
Pye JL, Garcia TC, Kapatkin AS, Samol MA, Stover S.To compare (1) performance of compact versus standard flute drill bits, (2) screw insertion properties and (3) pullout variables between interlocking thread (ITS) and buttress thread (BTS) self-tapping screws in third metacarpi. Methods: In vitro experimental study. Methods: Paired third metacarpi from 11 Thoroughbreds aged 2-4 years. Methods: Screws were inserted into the lateral condylar fossae following bone preparation using the respective drill bit for each screw type. Screw pullout was achieved using a mechanical testing system. Density and porosity of bone surrounding screw holes was ...
Wilkołek P, Szczepanik M, Sitkowski W, Rodzik B, Pluta M, Taszkun I, Gołyński M.Although intradermal testing (IDT) is commonly used in the etiological diagnosis of allergies, in vitro testing for specific IgE (sIgE) is an attractive alternative. Currently, new laboratory techniques in veterinary allergological practice, including multiple allergen simultaneous tests (MASTs), gradually supersede in vivo tests. Both, serological (sIgE) and IDTs in fourteen atopic Malopolski horses were performed. Correlation and agreement between test results were evaluated. Receiver operating characteristic analysis showed that sIgE to had the best diagnostic performance (Area under the R...
Koneberg DG, Edinger J.The purpose of this in vitro study was to evaluate the value of three-dimensional (3D) ultrasonography for the diagnosis of equine meniscal and trochlear ridge lesions under in vitro conditions. Lesions were created in the isolated meniscus and femoral trochlea of 25 cadaver stifle joints. Cylindric, conic, and cuboid lesions were created on the trochlear ridge. Five different meniscal tear configurations were created. A total of 107 lesions of the trochlear ridge and 103 lesions of the meniscus were created. 3D ultrasonography was performed in a waterbath, using a 7.5 MHz 3D scanner. Trochlea...
Sun Y, Chen H, Kandel R, Hurtig M.Lesions in cartilage of equine weightbearing joints commonly result in lameness. Cell-based resurfacing techniques are currently being developed for human and veterinary applications. Biopsies of stifle joint cartilage (1 g) were harvested aseptically and chondrocytes were isolated by sequential enzyme digestion. The cells were grown in vitro on filter inserts. Analysis of cultures 8 weeks later showed that the cells had accumulated extracellular matrix and formed a continuous layer of cartilagenous tissue as determined histologically. The cells maintained their phenotype as they synthesised t...
Wilson DA, Keegan KG, Carson WL.This study compared the mechanical properties of the normal intact suspensory apparatus and two methods of fixation for repair of transverse, midbody fractures of the proximal sesamoid bones of adult horses: transfixation wiring (TW) and screws placed in lag fashion (LS). Methods: An in vitro, paired study using equine cadaver limbs mounted in a loading apparatus was used to test the mechanical properties of TW and LS. Methods: Seventeen paired (13 repaired, 4 normal) equine cadaver limbs consisting of the suspensory apparatus third metacarpal bone, and first and second phalanges. Methods: The...
Bertram CA, Dietert K, Pieper L, Erickson NA, Barton AK, Klopfleisch R.During bronchoalveolar lavage fluid (BALF) sample preparation in horses, several technical aspects can affect sample variability. To date, the effects of different fixatives on prepared equine BALF films have been insufficiently investigated. Objective: This study aimed to determine the effect of various on-slide fixation methods on cell quality, including spray fixation of wet films, and acetone and methanol fixation of air-dried samples in comparison with unfixed, air-dried films. Methods: Cytocentrifuged BALF samples from 5 horses were fixed in a wet state using a commercially available fix...
Ball MA, Rebhun WC, Trepanier L, Gaarder J, Schwark WS.The objectives of this study were to determine the concentration of itraconazole achieved in corneal tissue and aqueous humour after topical application of a 1% itraconazole ointment: to determine the effect of including dimethyl sulphoxide (DMSO) in the ointment on achievable ocular tissue itraconazole concentrations; and to assess if any gross or histopathologic ocular toxicity results from the topical application of 1% itraconazole with or without the addition of DMSO. The experimental trial consisted of 6 horses considered to have normal eyes. Each horse had one eye treated with 0.3 mL of ...
Ho EN, Kwok WH, Leung DK, Wan TS, Wong AS.Turinabol (4-chloro-17alpha-methyl-17beta-hydroxy-1,4-androstadien-3-one) is a synthetic oral anabolic androgenic steroid. As in the case of other anabolic steroids, it is a prohibited substance in equine sports. The metabolism of turinabol in human has been reported previously; however, little is known about its metabolic fate in horses. This paper describes the studies of both the in vitro and in vivo metabolism of turinabol in racehorses with an objective to identify the most appropriate target metabolites for detecting turinabol administration. For the in vitro studies, turinabol was incub...
Milenkovic D, Mata X, Chadi S, Guérin G.Laminins are large heterotrimeric basement membrane glycoproteins composed of alpha, beta and gamma chains. The Laminin 5 isoform has an alpha3beta3gamma2 composition and is essential for the adhesion of basal keratinocytes to the underlying epithelial basement membrane where it is mainly located. Mutations in the genes coding for the 3 chains have been associated with a severe skin blistering disease, Herlitz's junctional epidermolysis bullosa (JEB), observed in different species as man, dog, cat and horse. In this study, we report the sequence of the 5.2 kb horse laminin alpha 3 cDNA (LAMA3)...
MacKay RJ, Wyer S, Gilmour A, Kongara K, Harding DR, Clark S, Mayhew IG, Thomson CE.Pasture-associated stringhalt is an acquired equine disease characterized by peripheral neuropathy and hyperflexion of the pelvic limbs. The disease occurs most commonly during periods of drought in horses grazing pastures heavily contaminated by Hypochaeris radicata. We hypothesized that stringhalt is caused by neurotoxins elaborated by H. radicata in response to the stress of drought conditions. Supernates were collected from H. radicata that were stressed (or not) by immersion in copper chloride solution, then extracted with ethyl acetate and dried. Dilutions of extracts from stressed (SE) ...
Han X, Quan Y, Gao X, Xiang W, Zhou J.To elucidate the role of N-glycosylation in fetal donkey dermal cell (FDD)-attenuated equine infectious anemia virus (EIAV), we constructed an N-glycosylation reverse-mutation molecular clone, pLGN191N236N246. This viral molecular clone was derived from the infectious clone pLGFD3-8 by site-directed mutagenesis. This clone was used to transfect fetal donkey dermal (FDD) cells. Infectious characteristics of transfectants were monitored by RT-PCR, indirect immune fluorescence and reverse transcriptase activity assay. After three passages in FDD cells, viral replications in the supernatant of cel...
McInturff C, Zanotto G, McInturff M, Hendrickson D.Injection of the equine navicular bursa can be technically challenging, and inadvertent penetration of other synovial structures is common using previously described techniques. When injecting the navicular bursa, inadvertent penetration of other synovial structures and hoof configuration can affect success rate, especially when performed by inexperienced operators. The aim of this study is to describe an alternate radiographic guided technique for injection of the equine navicular bursa that consistently avoids penetration of the distal interphalangeal joint using a 40 mm (1.5-inch) 20-gauge ...
Burton AG, Clark KC, Borjesson DL, Carrade DD, Burges J, Owens SD.Volume reduction and RBC depletion of equine bone marrow specimens are necessary processing steps for the immediate therapeutic use of bone marrow (BM)-derived mesenchymal stem cells (MSC), and for MSC expansion in culture. Objective: The purpose of the study was to evaluate the ability of the PrepaCyte-CB processing system to reduce volume, deplete RBC, and recover mononuclear cells (MNC) from equine BM specimens. Methods: One hundred and twenty mL of heparinized BM were obtained from each of 90 horses. A CBC was performed on the BM pre- and post-PrepaCyte-CB processing. Volume and RBC reduct...
Whitlock D, Garcia TC, Vallance SA, Stover SM.The specific biomechanical circumstances that induce excessive superficial digital flexor tendon (SDFT) strain in horses are unknown. Objective: Carpal joint hyperextension during axial limb loading during the middle of stance disproportionately enhances SDFT strains compared to suspensory ligament (SL) strains. Methods: Superficial digital flexor tendon and SL strains were measured in 7 cadaver limbs during in vitro loading that maintained carpal extension or allowed carpal hyperextension by constraining, or allowing rotation of, the radius during loading conditions that simulated the middle ...
Kolmakova MV, Kuskova ZR, Ratner GM, Laptakova LM.Horse serum albumin has been shown to meet the requirements to protein preparations for microanalysis and thus to be suitable for use in kits of reagents for the radioimmunological determination of insulin and myoglobin, for the determination of tick-borne encephalitis virus antigen by the method of the enzyme immunoassay and for the stabilization of proteins in the hemagglutination test and the hemagglutination inhibition test.
Word TA, Larsen RW.Here the molar volume and enthalpy changes associated with the early events in the folding of ferrocytochrome c (Cc) at high pH have been examined using time resolved photoacoustic calorimetry (PAC). The data reveal an overall volume change of 1.3 ± 0.3 mL mol and an enthalpy change of 13 ± 7 kcal mol occurring subsequent to photodissociation of the unfolded CO bound Cc species in <∼20 ns. Two additional kinetic phases are observed that are associated with non-native His binding (ΔH and ΔV of 2 ± 4 kcal mol and -0.5 mL mol, τ ∼ 2.5 μs ) and Met binding (ΔH an...
Marsan C, Goff AK, Sirois J, Betteridge KJ.Horse conceptuses were recovered non-surgically at Day 12-Day 15 and were dissociated with collagenase. Separation of the cells on a 31.8% Percoll gradient gave two bands of cells and indirect evidence suggests that the low density cells (LDC) are endoderm and the higher density cells (HDC) are trophectoderm. Each band was incubated for 24 h in Minimum Essential Medium and concentrations of oestradiol and progesterone in the medium were measured by radioimmunoassay (RIA). The LDC secreted predominately progesterone (log oestradiol/progesterone = -0.994 +/- 0.141; N = 15) whereas the HDC secret...
