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.
Wattrang E, Berg M, Magnusson M.This study aimed to evaluate different nucleic acid preparations as cytokine inducers in equine cells. To induce cytokine production, bacterial plasmid DNA or short synthetic oligodeoxyribonucleotides (ODN), with or without the transfection reagent lipofectin, were added to cultures of purified equine peripheral blood mononuclear cells (PBMC). Cytokine activity was detected with bioassays in cell culture supernatants after 24h of induction and cytokine mRNA expression was detected using RT-PCR at 6h post induction. For IFN-alpha/beta it was found that both plasmid DNA and phosphodiester ODN, c...
Al Jassim RA, Scott PT, Trebbin AL, Trott D, Pollitt CC.Seventy-two lactic acid producing bacterial isolates (excluding streptococci) were cultured from the gastrointestinal tract of six horses. Two of the horses were orally dosed with raftilose to induce lactic acidosis and laminitis while the remaining four were maintained on a roughage diet. Near complete 16S rDNA was amplified by PCR from the genomic DNA of each isolate. Following RFLP analysis with the restriction enzymes MboI, HhaI and HinfI, the PCR products from the 18 isolates that produced L- and/or D-lactate were subsequently cloned and sequenced. DNA sequence analysis indicated that the...
Claes F, Ilgekbayeva GD, Verloo D, Saidouldin TS, Geerts S, Buscher P, Goddeeris BM.In this study, we compared the complement fixation test (CFT), the horse complement fixation test (HCFT) and a card agglutination test for trypanosomosis (CATT/T. evansi) for the diagnosis of equine trypanosomosis in the Republic of Kazakhstan. Cohen's kappa test was used to evaluate the concordance between the three tests. Kappa scores for CFT versus HCFT and CATT are both 0.6165 (95% Confidence Interval CI 0.414--0.819) indicating a "substantial" agreement between CFT and HCFT or CATT, respectively. Kappa for HCFT versus CATT is 0.395 (CI 0.142--0.648) indicating a "fair" agreement between t...
Jacobsen S, Kjelgaard-Hansen M, Hagbard Petersen H, Jensen AL.The aim of the present study was to evaluate whether equine serum amyloid A (SAA) concentrations could be measured reliably with a turbidometric immunoassay (TIA) developed for use with human serum. Intra- and inter-assay imprecision were evaluated by multiple measurements on equine serum pools. Assay inaccuracy was determined by linearity under dilution. The assay was subsequently used for measuring SAA concentrations in clinically healthy horses, horses with inflammatory diseases, horses with non-inflammatory diseases, and in horses before and after castration. In pools with low, intermediat...
Neild DM, Brouwers JF, Colenbrander B, Agüero A, Gadella BM.In this study we used a new method to detect reactive oxygen species (ROS) induced damage at the level of the sperm plasma membrane in fresh and frozen-thawed stallion sperm. Lipid peroxidation (LPO) in sperm cells was assessed by a fluorescent assay involving the labeling of stallion sperm with the LPO reporter probe C11-BODIPY(581/591). The peroxidation dependent spectral emission shift of this membrane probe could be localized using inverted spectral confocal microscopy and quantified on living and deteriorated sperm cells using flow cytometry. Mass spectrometric analysis of the main endoge...
Rambags BP, Krijtenburg PJ, Drie HF, Lazzari G, Galli C, Pearson PL, Colenbrander B, Stout TA.Chromosomal aberrations are often listed as a significant cause of early embryonic death in the mare, despite the absence of any concrete evidence for their involvement. The current study aimed to validate fluorescent in situ hybridization (FISH) probes to label specific equine chromosomes (ECA2 and ECA4) in interphase nuclei and thereby determine whether numerical chromosome abnormalities occur in horse embryos produced either in vivo (n = 22) or in vitro (IVP: n = 20). Overall, 75% of 36,720 and 88% of 2,978 nuclei in the in vivo developed and IVP embryos were analyzable. Using a scoring sys...
Shaw FL, Handy RD, Bryson P, Sneyd JR, Moody AJ.The aim of the study was to investigate whether a single hyperbaric oxygen exposure causes oxidative stress in isolated platelets. Methods: Isolated horse platelets were exposed to 100% oxygen at 2.2 atmospheres, or 100% oxygen under normobaric conditions, or air under normobaric conditions for 90 min. Results: There were no differences in platelet SOD activity between conditions, but there was a rise in SOD in all cases after 24 h (in control platelets at 24 h, SOD was 11.9 +/- 1.9 nmol/min/mg protein compared to initial background levels of 8.2 +/- 1.9 nmol/min/mg protein) (P < 0.05). Nei...
Harrington JR, Golding MC, Martens RJ, Halbert ND, Cohen ND.To evaluate a real-time quantitative polymerase chain reaction (QPCR) assay in the detection and quantitation of virulent Rhodococcus equi. Methods: 1 virulent, 2 intermediately virulent, and 2 avirulent strains of R. equi and 16 isolates of bacteria genetically related to R. equi. Methods: The QPCR assay was evaluated for detection and quantitation of the virulence-associated gene (vapA) of R. equi in pure culture and in samples of tracheobronchial fluid, which were inoculated with known numbers of virulent R. equi. Results were compared with those derived via quantitative microbial culture a...
Estepa JC, Lopez I, Mayer-Valor R, Rodriguez M, Aguilera-Tejero E.The aim of this study was to evaluate the influence of two commonly used anticoagulants (K3EDTA and lithium heparin) on refractometric and spectrophotometric measurement of total protein (TP) concentration in equine peritoneal fluid samples. The influence of a commercial solution of K3EDTA, a solution of K3EDTA in distilled water and lithium heparin on the refractometric and spectrophotometric (biuret) quantification of TP content in peritoneal fluid samples was assessed. Total protein concentration measured by refractometry was consistently overestimated in samples with commercial K3EDTA. The...
Hartley CA, Wilks CR, Studdert MJ, Gilkerson JR.To compare methods of detecting equine herpesvirus type 1 (EHV1)- and EHV4-specific antibodies in horse sera. Methods: 33 acute and convalescent serum samples from experimentally or naturally infected horses after confirmed EHV1 or EHV4 infection. Methods: For each sample, serum antibody titers against EHV1 and EHV4 were determined by use of virus neutralization (VN) and complement fixation (CF) assays. The ELISA absorbance values for each serum sample were determined against the EHV1 and EHV4 recombinant ELISA antigens. Values obtained for acute and convalescent sera in each assay were compar...
Szeredi L, Hornyák A, Pálfi V, Molnár T, Glávits R, Dénes B.The occurrence of equine arteritis virus (EAV) induced equine abortions was studied with different laboratory methods during a 3-year period. Tissue samples from 96 aborted equine foetuses or newborn foals were collected from 57 farms located in different parts of Hungary. Virus isolation, polymerase chain reaction (PCR), immunohistochemistry and serology were used for the detection of EAV infection. The overall seroprevalence of EAV infection in mares was 65%. EAV induced abortion was diagnosed in eight (8.3%) cases from six (10.5%) herds. Abortion was sporadic in all herds except for one, wh...
Henson FM, Bowe EA, Davies ME.To identify the effect of fibroblastic growth factor-2 (FGF-2) on the intrinsic damage-repair response in articular cartilage in vitro. Methods: Articular equine cartilage explants, without subchondral bone, had a single impact load of 500 g applied from a height of 2.5 cm. Explants were then cultured in 0, 12, 25, 50 or 100 ng/ml FGF-2 for up to 28 days. Unimpacted discs served as controls for each time-point. Histological and immunohistochemical techniques were used to quantify and characterise the response of putative chondrocyte progenitor cells (CPC) to damage and FGF-2 treatment. Results...
Soma LR, Uboh CE, Guan F, Luo Y, Moate PJ, Boston RC, Driessen B.Hemoglobin-glutamer-200 (HBOC-200) is a hemoglobin (Hb)-based oxygen carrier (HBOC) comprising glutaraldehyde-polymerized bovine Hb. In this study, we sought to determine the pharmacokinetics of this first generation HBOC after IV infusion of 32.5 g of HBOC-200 solution in horses. Quantification of HBOC-200 in equine plasma and urine was performed using a method recently developed by our laboratory. The elimination from plasma was based on size distribution of the bovine Hb polymer. The decline of plasma concentration-time curve of HBOC-200 was described by a noninterchanging 2-compartmental m...
Zapata GL, Britos RM, Pintos ME, Dreizzen E, Lausada NB, Arauz S.The objective of this paper was to determine the physiological values of urea nitrogen and creatinine in tears, and to compare the results with those obtained from serum. Thirty healthy thoroughbred horses were included in the study. Tear fluid samples were obtained using a glass capillary tube placed in lower conjunctival cul-de-sac. Blood samples were taken from the jugular vein. Tear and serum urea nitrogen and creatinine levels were quantitatively analyzed by an enzymatic colorimetric method. Urea nitrogen values were 4.22+/-1.84 mmol/l in tears and 4.44+/-1.78 mmol/l in serum, whereas cre...
The measurement of cell proliferation and cell viability using 5'bromo-2'deoxy-uridine (BrdU) labelling has been described in several cell types and species. The aim of this study was to adapt this technique to equine embryos and to compare the index of DNA replication (S-phase) between equine and caprine embryos. Seventeen equine embryos were recovered at day 6.5 post-ovulation and 20 caprine embryos were recovered at day 7 after the onset of estrus. Equine embryos were incubated during 1h at 39 degrees C in PBS containing 1mM of BrdU. Embryos were then treated in 0.05% trypsin during 15 min ...
Steffey EP, Mama KR, Galey FD, Puschner B, Woliner MJ.To quantitate effects of dose of sevoflurane and mode of ventilation on cardiovascular and respiratory function in horses and identify changes in serum biochemical values associated with sevoflurane anesthesia. Methods: 6 healthy adult horses. Methods: Horses were anesthetized twice: first, to determine the minimum alveolar concentration (MAC) of sevoflurane and second, to characterize cardiopulmonary and serum biochemical responses of horses to 1.0, 1.5, and 1.75 MAC multiples of sevoflurane during controlled and spontaneous ventilation. Results-Mean (+/- SEM) MAC of sevoflurane was 2.84 +/- ...
Kon H, Tohei A, Hokao R, Shinoda M.The estrous cycle influence on the number of ovulated eggs after injection of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) was investigated in 12, 18, and 24 weeks old adult female Wistar-Imamichi (WI) rats. PMSG (150 IU/kg) was injected at metestrus, diestrus, proestrus, or estrus, followed by hCG (75 IU/kg) 55 h later. Ovulation was induced at all ages and stages of the estrous cycle. The number of ovulated eggs was not affected by stage for similarly aged rats, however, the number of ovulated eggs obtained after treatment decreased with age. These results d...
Zdovc I, Ocepek M, Gruntar I, Pate M, Klobucar I, Krt B.The prevalence of Taylorella equigenitalis infection in Slovenia is unknown and methods used to refine identification in these stallions are required. Objective: In diagnosis of T. equigenitalis, polymerase chain reaction (PCR) would have advantages over culture methods, especially in cases where small numbers of causal agent or intensive contamination of genital swabs are involved. Methods: Culture method and PCR were used to examine a total of 980 genital swabs from the urethra and fossa urethralis of 245 stallions for the presence of the contagious equine metritis organism. Results: Among 2...
Sankaranarayanan R, Biswal BK, Vijayan M.A new relaxed state has been characterized in the crystals of horse methemoglobin grown at neutral pH at low ionic concentration and their low humidity variants. The crystals provide an example for improvement in X-ray diffraction quality with reduced solvent content. Only the classical R state has been so far observed in liganded horse hemoglobin. The state characterized in the present study lies in between the R state and the R2 state characterized earlier in liganded human hemoglobin. The results presented here, along with those of earlier studies, suggest that relaxed and tense hemoglobin ...
Cook RF, Cook SJ, Bolin PS, Howe LJ, Zhou W, Montelaro RC, Issel CJ.In the context of DNA vaccines the native equine infectious anemia virus (EIAV)-envelope gene has proven to be an extremely weak immunogen in horses probably because the RNA transcripts are poorly expressed owing to an unusual codon-usage bias, the possession of multiple RNA splice sites and potential adenosine-rich RNA instability elements. To overcome these problems a synthetic version of sequences encoding the EIAV surface unit (SU) envelope glycoprotein was produced (SYNSU) in which the codon-usage bias was modified to conform to that of highly expressed horse and human genes. In transfect...
Sepulveda MF, Greenaway EC, Avella M, Goode NT, Cunningham FM.To determine if protein kinase C (PKC) regulates equine eosinophil function. Methods: Blood eosinophils were obtained from healthy ponies. Methods: IL-5- and histamine-induced adherence to serum-coated plastic was measured as the eosinophil peroxidase content of adherent cells and serum treated zymosan (STZ)-and IL-5-induced superoxide production by the reduction of cytochrome C. Eosinophil PKC activity was quantitated as the rate of transfer of (32)P from ATP to substrate. The effects of Ro31-8220 (isotype non-selective PKC inhibitor), Go6976 (conventional PKC inhibitor), and rottlerin (PKCde...
Spencer JA, Deinnocentes P, Moyana EM, Guarino AJ, Ellison SE, Bird RC, Blagburn BL.Equine protozoal myeloencephalitis (EPM) is a neurologic syndrome seen in horses from the Americas and is mainly caused by Sarcocystis neurona. Recently, a 29-kDa surface antigen from S. neurona merozoites was identified as being highly immunodominant on a Western blot. This antigen has been sequenced and cloned, and the expressed protein has been named SnSAG1. In a previous study, cell-mediated immune responses to SnSAG1 were shown to be statistically significantly reduced in horses with EPM in comparison to EPM-negative control horses. It therefore appears as though the parasite is able to i...
Yu NH, Ho EN, Leung DK, Wan TS.Anabolic steroids have the capability of improving athletic performance and are banned substances in the Olympic games as well as in horseracing and equestrian competitions. The control of their abuse in racehorses is traditionally performed by detecting the presence of anabolic steroids and/or their metabolite(s) in urine samples using gas chromatography-mass spectrometry (GC-MS). However, this approach usually requires tedious sample processing and chemical derivatisation steps and could be very insensitive in detecting certain steroids. This paper describes a high performance liquid chromat...
Morris MD, Matousek P, Towrie M, Parker AW, Goodship AE, Draper ER.Picosecond time-resolved Raman spectroscopy in equine cortical bone tissue is demonstrated. Using 400-nm pulsed laser excitation (1 ps at 1 kHz) it is shown that Kerr cell gating with a 4-ps window provides simultaneously time-resolved rejection of fluorescence and time-resolved Raman scatter enabling depth profiling through tissue. The Raman shifts are the same as those observed by conventional cw Raman spectroscopy using deep-red or near-infrared lasers. The time decay of Raman photons is shown to fit an inverse square root of time function, suggesting propagation by a diffusive mechanism. U...
Luo Y, Uboh CE, Soma LR, Guan FY, Rudy JA, Tsang DS.A method for the simultaneous separation, identification, quantification and confirmation of the presence of 21 glucocorticoids (GCC) in equine plasma by liquid chromatography coupled with triple stage quadrupole tandem mass spectrometry (LC/TSQ-MS/MS) is described. Plasma sample augmented with the 21 GCC was extracted with methyl tert-butyl ether (MTBE) and analyzed by positive electrospray ionization. Desoxymetasone or dichlorisone acetate was used as the internal standard (IS). Quantification was performed by IS calibration. For each drug, one major product ion was chosen and used for scree...
Traversa D, Otranto D, Iorio R, Giangaspero A.Equine thelaziosis caused by the eyeworm Thelazia lacrymalis is a parasitic disease transmitted by muscid flies. Although equine thelaziosis is known to have worldwide distribution, information on the epidemiology and presence of the intermediate hosts of T. lacrymalis is lacking. In the present work, a PCR-RFLP based assay on the first and/or second internal transcribed spacer (ITS1 and ITS2) of ribosomal DNA was developed for the detection of T. lacrymalis DNA in its putative vector(s). The sensitivity of the technique was also assessed. The restriction patterns obtained readily differentiat...
Pitzer JB, Kaufman PE, Geden CJ, Hogsette JA.The ability of Spalangia cameroni Perkins, Spalangia endius Walker, and Muscidifurax raptorellus Kogan and Legner to locate and attack stable fly hosts was evaluated under laboratory conditions. Postfeeding third-instar stable fly larvae were released and allowed to pupate in two arena types: large 4.8 liter chambers containing a field-collected, soiled equine bedding substrate; or 120-ml plastic cups containing wood chips. At the time of fly pupariation, parasitoids were released and permitted 72 h to locate and attack hosts. On average, parasitism rates of freely accessible stable fly pupae ...
Donofrio JC, Coonrod JD, Chambers TM.Influenza A is a common respiratory infection of horses, and rapid diagnosis is important for its detection and control. Sensitive detection of influenza currently requires viral culture and is not always feasible. The polymerase chain reaction (PCR) was used to detect DNA produced by reverse transcription of equine influenza in stored nasal secretions, vaccines, and allantoic fluids. Primers directed at a target of 212 bp on conserved segment 7 (matrix gene) of human influenza A/Bangkok/1/79(H3N2) produced amplification products of appropriate size with influenza A/Equine/Prague/1/56 (H7N7), ...
Verma RD, Venkateswaran KS, Sharma JK, Agarwal GS.Malleo-proteins from synthetic broth mallein of six strains of Pseudomonas mallei were separated by trichloroacetic acid precipitation, ammonium sulfate precipitation and Ultrogel AcA 34 gel filtration chromatography. When tested comparatively with Dutch PPD mallein as standard on P. mallei-sensitized and normal horses all the strains were found to be malleinogenic, trichloroacetic acid precipitated proteins were comparable to Dutch PPD mallein in potency and innocuity whereas ammonium sulfate-precipitated proteins elicited non-specific reactions. Ultrogel AcA 34 chromatographed high molecular...
Zhang H, Nagaoka K, Imakawa K, Nambo Y, Watanabe G, Taya K, Weng Q.The establishment of equine pregnancy is a unique and long process during which a series of physical and possibly biochemical interactions are required between the conceptus and uterus. In this study, we investigated the expression pattern of inhibin/activin subunits in the uterus during early pregnancy. The uteri from four adult mares on cyclic day 13 or pregnancy day 25 were obtained. Immunohistochemical experiments suggested that inhibin/activin subunits were immunolocalized in the luminal and glandular epithelium on pregnancy day 25. In addition, the inhibin α and inhibin/activin βB subu...
Inkerman PA, Scott K, Runnegar MT, Hamilton SE, Bennett EA, Zerner B.Chicken, sheep, and horse liver carboxylesterases have been purified by procedures involving ammonium sulfate fractionation, ion-exchange chromatography and gel filtration on Sephadex. The actual yields of the procedures described were as follows: chicken, 1 g from 2 kg of liver powder (chloroform-acetone); sheep, 200 mg from 400 g of powder (chloroform-acetone); horse, 230 mg from 800 g of powder (acetone). The purified enzymes are free of non-carboxyl-esterase protein as shown by gel electrophoresis, although they do contain electrophoretic variants. The equivalent weight of the chicken enzy...
Sampaio B, Ortiz I, Resende H, Felix M, Varner D, Hinrichs K.Exposure to the calcium ionophore A23187 may present a "universal" sperm treatment for IVF, as it bypasses capacitation pathways. However, success in utilizing A23187 is variable, especially in equine spermatozoa. Notably, albumin is used during A23187 treatment but paradoxically is thought to suppress A23187 action. Essentially no critical data are available on the effects of A23187 and albumin concentrations, ratios, or addition protocols on changes in intracellular calcium ([Ca] ) in any cell type. To determine factors that affect the action of A23187 on [Ca] in equine and murine spermatozo...
Schambony A, Hess O, Gentzel M, Töpfer-Petersen E.Cysteine rich secretory proteins (CRISPs) have been detected immunochemically in the equine male genital tract. CRISPs are secretory products of the epididymis, the ampulla and the seminal vesicle. A particular feature of the horse is the abundance of CRISPs in seminal plasma. CRISPs can also be detected in extracts of testicular, epididymal and ejaculated spermatozoa in increasing amounts. Unlike other seminal plasma proteins, they cannot be removed completely from spermatozoa by high salt treatment. The remaining CRISP antigens are localized on the midpiece, and the postacrosomal and equator...
Hutchins JE, Tyczkowska K, Aronson AL.A new liquid chromatographic method for determination of ampicillin in canine and equine serum has been developed. The serum sample (500 microL) is vortex-mixed with 20% ethanol (500 microL) and filtered using a 30,000 molecular weight cutoff microseparation tube to separate high molecular weight solutes following low-speed centrifugation. Ampicillin is then separated from other serum components by reverse phase ion-pair liquid chromatography (LC). The ultraviolet (UV) absorbance of the column effluent is monitored at 230 nm. Recoveries of ampicillin from canine serum spiked at concentrations ...
Carter RA, Ericsson SA, Corn CD, Weyerts PR, Dart MG, Escue SG, Mesta J.The objective of this study was to determine if spermatozoal reduction of the dyes methylene green to colorless and resazurin to pink or colorless was associated with the fertility potential of an equine semen sample. Fifty samples from 38 stallions were evaluated for the number of spermatozoa per milliliter and number of motile sperm per milliliter. Methylene green (20 micrograms/mL of semen) or resazurin (85 micrograms/mL of semen) was added to 3-mL aliquots of semen. Semen samples were identified as having low fertility potential (< 200 x 10(6) total cells/mL and or = 200 x 10(6) total ...
Benamou AE, Marlin DJ, Callingham BC, Hiley RC, Lekeux R.There is currently little published information about the effects of endothelin-1 (ET-1), a potent endogenous spasmogen of vascular and airway smooth muscle, on pulmonary vasculature and airways or which ET receptor subtypes mediate ET-1-induced vasoconstrictive and bronchoconstrictive action in the horse. Objective: To investigate the effect of endothelin-1 (ET-1) on smooth muscle from isolated equine pulmonary artery and bronchus. In addition, the roles of ETA and ETB receptors in ET-1 mediated contraction in these tissues were assessed. Methods: The force generation of ring segments from pu...
Noé B, Poole AR, Mort JS, Richard H, Beauchamp G, Laverty S.Develop a species-specific ELISA for a neo-epitope generated by cathepsin K cleavage of equine type II collagen to: (1) measure cartilage type II collagen degradation by cathepsin K in vitro, (2) identify cytokines that upregulate cathepsin K expression and (3) compare cathepsin K with matrix metalloproteinase (MMP) collagenase activity in stimulated cartilage explants and freshly isolated normal and osteoarthritic (OA) articular cartilages. A new ELISA (C2K77) was developed and tested by measuring the activity of exogenous cathepsin K on equine articular cartilage explants. The ELISA was the...
Subbiah M, Thirumalapura N, Thompson D, Kuchipudi SV, Jayarao B, Tewari D.Metagenomic sequencing of clinical diagnostic specimens has a potential for unbiased detection of infectious agents, diagnosis of polymicrobial infections and discovery of emerging pathogens. Herein, next generation sequencing (NGS)-based metagenomic approach was used to investigate the cause of illness in a subset of horses recruited for a tick-borne disease surveillance study during 2017-2019. Blood samples collected from 10 horses with suspected tick-borne infection and five apparently healthy horses were subjected to metagenomic analysis. Total genomic DNA extracted from the blood samples ...
Gray BP, Biddle S, Pearce CM, Hillyer L.Fluticasone propionate (FP) is an anti-inflammatory agent with topical and inhaled applications commonly used in the treatment of asthma in steroid-dependent individuals. The drug is used in racehorses to treat Inflammatory Airway Disease; this work was performed in order to advise on its use and detect potential misuse close to racing. Methods were developed for the extraction and analysis of FP from horse plasma and a carboxylic acid metabolite (FP-17βCOOH) from horse urine. The methods utilize ultra high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) in ...
Pellegrini A, von Fellenberg R.The principal alpha-1-protease inhibitor of horse was fractionated by classical methods and analysed with a modified fibrinogen-agarose gel electrophoretic method of high sensitivity and resolving power. Starting with an electrophoretically homogeneous inhibitor in unfractionated serum, two isoinhibitor bands became apparent after fractionation with (NH4)2SO4 and DEAE-cellulose DE-52 ion-exchange chromatography. The isoinhibitors differed in electrophoretic migration and in the elution pattern from Sephadex G-100 gel filtration, but possessed identical antigenic determinants and enzyme specifi...
Piccione G, Assenza A, Grasso F, Caola G.BACKGROUND: Many physiological processes of mammalian species exhibit daily rhythmicity. An intrinsic relationship exists between fat soluble vitamins (A, D, E and K) and several body functions. Few investigations on the rhythmic pattern of vitamins in domestic animals have been carried out. The present study evaluated the circadian rhythmicity of fat soluble vitamins in the horse. METHODS: Blood samples from 5 Thoroughbred mares were collected at four-hour intervals over a 48-hour period (starting at 8:00 hours on day 1 and finishing at 4:00 on day 2) via an intravenous cannula inserted into ...
Grace PB, Drake EC, Teale P, Houghton E.Following administration of the anabolic steroid 19-nortestosterone or its esters to the horse, a major urinary metabolite is 19-nortestosterone-17beta-sulphate. The detection of 19-nortestosterone in urine from untreated animals has led to it being considered a naturally occurring steroid in the male horse. Recently, we have demonstrated that the majority of the 19-nortestosterone found in extracts of 'normal' urine from male horses arises as an artefact through decarboxylation of the 19-carboxylic acid of testosterone. The aim of this investigation was to establish if direct analysis of 19-n...
Wong AS, Ho EN, Wan TS, Lam KK, Stewart BD.Bisphosphonates are used in the management of skeletal disorder in humans and horses, with tiludronic acid being the first licensed veterinary medicine in the treatment of lameness associated with degenerative joint disease. Bisphosphonates are prohibited in horseracing according to Article 6 of the International Agreement on Breeding, Racing and Wagering (published by the International Federation of Horseracing Authorities). In order to control the use of bisphosphonates in equine sports, an effective method to detect the use of bisphosphonates is required. Bisphosphonates are difficult-to-de...
Kaabia Z, Dervilly-Pinel G, Hanganu F, Cesbron N, Bichon E, Popot MA, Bonnaire Y, Le Bizec B.During last decades, the use of natural steroids in racing and food producing animals for doping purposes has been flourishing. The endogenous or exogenous origin of these naturally occurring steroids has since remained a challenge for the different anti-doping laboratories. The administration of these substances to animals is usually made through an intra-muscular pathway with the steroid under its ester form for a higher bioavailability and a longer lasting effect. Detecting these steroid esters would provide an unequivocal proof of an exogenous administration of the considered naturally occ...
Jones B, Taylor K, Lucas RM, Merritt T, Chicken C, Heller J, Carrick J, Givney R, Durrheim DN.This report describes the challenges encountered in using serological methods to study the historical transmission risk of from horses to humans. In 2017, serology and risk factor questionnaire data from a group of individuals, whose occupations involved close contact with horses, were collected to assess the seroprevalence of antibodies to and identify risk factors associated with previous exposure. 147 participants were enrolled in the study, provided blood samples, and completed a questionnaire. On ELISA testing, antibodies to the Chlamydia genus were detected in samples from 17 participa...
Cehak A, Wilkens MR, Guschlbauer M, Mrochen N, Schröder B, Feige K, Breves G.Transepithelial transport mechanisms play a key role in regulating the absorption and secretion of calcium (Ca(2+)) and inorganic phosphate (P(i)) in the gastrointestinal tract. Although intestinal disorders with imbalances in macromineral homeostasis are frequently observed in horses, available data on intestinal Ca(2+) and P(i) transport are limited. The aim of the present study was to characterize the intestinal Ca(2+) and P(i) transport functionally by using the in vitro radioisotope tracer technique with Ussing chambers and to identify components involved in Ca(2+) transport at both mRNA ...
Hormanski CE, Truax R, Pourciau SS, Folsom RW, Horohov DW.The in vitro stimulation of peripheral blood mononuclear cells (PBMC) with interleukin 2 (IL-2) results in the development of potent cytotoxic effector cells, referred to as lymphokine-activated killer (LAK) cells. LAK cells are capable of lysing a wide variety of autologous, allogeneic and xenogeneic tumor cells. The exact mechanism of target cell recognition by LAK cells remains unknown. LAK cell activity has been reported for a variety of domesticated species except the horse. We report here that IL-2-stimulated equine PBMC, which fail to lyse either human or murine tumor cell lines, exhibi...
Camp CJ, Leid RW.A method for the isolation of equine neutrophils was developed using metrizamide cushions. A purity of greater than 95% was routinely obtained with greater than 90% viability. These cells were radiolabeled and tested for their chemotactic response in Boyden chambers to zymosan-activated equine serum, the partially purified equine complement component C5a, and formyl-L-methionyl-L-leucyl-L-phenylalanine. The time and ionic requirements for chemotaxis of radiolabeled equine neutrophils were investigated and maximal movement was observed at 2 hours' incubation and 1.0 mM Ca and 0.5 mM Mg. Dinitro...
Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota K, Kusano K, Nagata S.Gene doping is prohibited in horseracing. In a previous study, we developed a method for non-targeted transgene detection using DELLY, which is based on split-read (SR) and paired-end (PE) algorithms to detect structural variants, on WGS data. In this study, we validated the detection sensitivity of DELLY using artificially generated sequence data of 12 target genes. With DELLY, at least one intron was detected as a deletion in eight targeted genes using the 150 bp PE read WGS data, whereas all targeted genes were detected by DELLY using the 100 bp PE read data. The detection sensitivity was h...
Varner DD, Vaughan SD, Johnson L.Three ejaculates from each of 3 stallions were used to evaluate a computerized system (Hamilton-Thorn motility analyzer; HTMA) for measuring equine spermatozoal motility. Variance components (ejaculate-within-stallion, chamber-within-ejaculate, and microscopic field-within-chamber) were determined for each stallion after diluting ejaculates to 25 x 10(6) spermatozoa/ml with a skim milk-glucose seminal extender. The HTMA was compared with frame-by-frame playback videomicrography (VIDEO) for determining: percentage of spermatozoal motility and spermatozoal number in microscopic fields; curviline...
Kinsley R, Pronost S, De Bock M, Temperton N, Daly JM, Paillot R, Scott S.Equine influenza is a major respiratory disease of horses that is largely controlled by vaccination in some equine populations. Virus-neutralising antibodies, the mainstay of the protective immune response, are problematic in assaying for equine influenza virus, as most strains do not replicate efficiently in cell culture. Surrogate measures of protective antibody responses include the haemagglutination inhibition (HI) test and single radial haemolysis (SRH) assay. For this study, a pseudotyped virus, bearing an envelope containing the haemagglutinin (HA) from the Florida clade 2 equine influe...
Lindsay DS, Dubey JP.Equine protozoal myeloencephalitis (EPM) is a serious neurological disease of horses in the Americas. The apicomplexan protozoan most commonly associated with EPM is Sarcocystis neurona. A direct agglutination test (SAT) was developed to detect antibodies to S. neurona in experimentally infected animals. Merozoites of the SN6 strain of S. neurona collected from cell culture were used as antigen and 2-mercaptoethanol was added to the antigen suspension to destroy IgM antibodies when mixed with test sera. Mice fed sporocysts of S. speeri or S. falcatula-like sporocysts from opossums did not sero...
Andrade DGA, Cerri FM, Barbosa GVM, Basso RM, Takahira RK, Pantoja JCF, Oliveira-Filho JP, Borges AS.Analysis of the cerebrospinal fluid (CSF) is important as a complementary test in horses with neurologic diseases, and sequential analysis may provide information about the treatment response or evolution and quantitative measures of the CSF drug concentration during treatment. The aim of this study was to compare erythrocyte and nucleated cell counts and protein concentration in multiple CSF samples obtained sequentially from two different puncture sites in clinically healthy horses. Eight and 12 horses, with no evidence of neurologic disease, were subjected to CSF collection from the atlanto...
Troedsson MH, Liu IK.Undiluted uterine secretion was used to determine the concentration of total protein and the accumulated volume of uterine secretion after a bacterial inoculation in mares susceptible and resistant to chronic uterine infection (CUI). The uterus of 6 susceptible and 5 resistant mares was inoculated with 5 x 10(6) Streptococcus zooepidemicus on the third day of estrus. Using a tampon inserted in the uterus, secretions were sampled at 5, 12, 24, and 36 hours after inoculation, followed by intrauterine lavage with phosphate buffered saline solution. The concentration of protein was determined in t...
Walter J, Klein C, Wehrend A.Exfoliative endometrial cytology is an easy and valuable diagnostic tool for the detection of inflammatory processes of the uterus that correlates well to culture results. The practical use of this procedure is limited due to the time-consuming Papanicolaou-Shorr staining technique. In this study the suitability of the rapid eosin-thiazin staining for endometrial smears was investigated. Methods: Sample collection was carried out with a guarded culture swab (Knudsen-catheter) in 27 broodmares during routine gynaecological examination. Two smears were prepared from each collection. One was stai...
Austbø L, Røed KH, Dolvik NI, Skretting G.The aim of this study was to investigate genes for differential expression in cartilage of foals predisposed to osteochondrosis (OC). Tissue was sampled from the cranial part of the distal intermediate ridge of the tibia in the tarso-crural joint. Foals were considered predisposed to OC when parents had OC at the distal intermediate ridge of the tibia. RNA was isolated and subjected to arbitrarily primed PCR (RAP-PCR) followed by fingerprinting to screen for differentially expressed genes. By verification of results from the RAP-PCR fingerprint screening using real-time RT-PCR, we identified t...
