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.
Ousey JC, Palmer L, Cash RS, Grimes KJ, Fletcher AP, Barrelet A, Foote AK, Manning FM, Ricketts SW.Standard bacteriological methods for identifying Taylorella equigenitalis in cervical smears are time consuming. Therefore, a more rapid real-time PCR assay was evaluated for its suitability in screening swabs. Objective: To compare the results of a commercially available real-time PCR assay with routine microbiological culture for the identification of T. equigenitalis, the causative organism of contagious equine metritis, in equine genital swab samples, under 'field trial' conditions. Methods: Routine prebreeding genital swabs (n=2072) collected from Thoroughbred mares and stallions during 2...
Vasconcelos AB, Santana MA, Santos AM, Santoro MM, Lagares MA.Microscopy has been used in the routine evaluation of sperm metabolism. Nevertheless, it has limited capacity to preview male fertility. As calorimetry may be used to evaluate directly the metabolic activity of a biological system, the aim of this study was to use microcalorimetry as an additive method for sperm metabolism evaluation of cooled equine semen. Two ejaculates of four stallions were collected and motility, viability (eosin 3%) and membrane functional integrity (hyposmotic swelling test) of spermatozoa were evaluated. Sperm samples were processed following different protocols and th...
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...
Bouwman FG, van Ginneken MM, van der Kolk JH, van Breda E, Mariman EC.The aim of the study was to identify new biomarkers for acute tying-up in horses. Skeletal muscle biopsies were taken from 3 horses suffering from acute tying-up and 3 healthy horses. We performed 2D gel electrophoresis and mass spectrometry for identification of proteins that are differentially expressed in tying-up. 2D gel electrophoresis of skeletal muscle sequential extracts yielded more than 350 protein spots on each gel, of which 14 were differentially expressed more than two-fold (p<0.05). In-gel digestion followed by peptide mass fingerprinting enabled identification of three significa...
Scarth JP, Clarke AD, Teale P, Pearce CM.Effective detection of the abuse of androgenic-anabolic steroids in human and animal sports often requires knowledge of the drug's metabolism in order to target appropriate urinary metabolites. 'Designer' steroids are problematic since it is difficult to obtain ethical approval for in vivo metabolism studies due to a lack of a toxicological profile. In this study, the in vitro metabolism of estra-4,9-diene-3,17-dione is reported for the first time. This is also the first study comparing the metabolism of a designer steroid in the three major species subject to sport's doping control; namely th...
Lettry V, Kawasaki H, Sugaya K, Hosoya K, Takagi S, Okumura M.This study aimed to evaluate a system that identifies cartilage turn over and/or degradation through measurement of a new keratan sulfate (KS) epitope concentration in equine sera. Blood samples were collected from 30 horses, 1 (n = 15) and 2 year-olds (n = 15). Serum samples were analyzed for an epitope of keratan sulfate by 1/20/5D4 (KS5D4) and new epitopes of keratan sulfate using high sensitive keratan sulfate (HSKS), measured by two respective enzyme-linked immunosorbant assays (ELISAs). There was no correlation in serum concentration of KS evaluated using 5D4 and HSKS. Age had no signifi...
Schmitz A, Thormann W, Moessner L, Theurillat R, Helmja K, Mevissen M.Ketamine, an injectable anesthetic and analgesic consisting of a racemic mixture of S-and R-ketamine, is routinely used in veterinary and human medicine. Nevertheless, metabolism and pharmacokinetics of ketamine have not been characterized sufficiently in most animal species. An enantioselective CE assay for ketamine and its metabolites in microsomal preparations is described. Racemic ketamine was incubated with pooled microsomes from humans, horses and dogs over a 3 h time interval with frequent sample collection. CE data revealed that ketamine is metabolized enantioselectively to norketamine...
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) ...
Vonaparti A, Lyris E, Panderi I, Koupparis M, Georgakopoulos C.Levodopa and dopamine have been abused as performance-altering substances in horse racing. Urinary 3-methoxytyramine is used as an indicator of dopaminergic manipulation resulting from dopamine or levodopa administration and is prohibited with a urinary threshold of 4 microg mL(-1) (free and conjugated). A simple liquid chromatographic (LC)/mass spectrometric (MS) (LCMS) method was developed and validated for the quantification and identification of 3-methoxytyramine in equine urine. Sample preparation involved enzymatic hydrolysis and protein precipitation. Hydrophilic interaction liquid chro...
Alvarez I, Gutierrez G, Barrandeguy M, Trono K.The purpose of this study was to develop and evaluate a simple immunochromatographic lateral flow (ICLF) test for specific detection of Equine infectious anemia virus (EIAV) antibodies in equine sera. Viral recombinant p26 capsid protein (rp26) was used as the capture protein in the test line and as the detector reagent conjugated to colloidal gold. The performance of rp26-ICLF was evaluated, and the results obtained were compared with a commercially available agar gel immunodiffusion (AGID) test used as a standard of comparison according to international guidelines. The values obtained for co...
Bellone RR, Forsyth G, Leeb T, Archer S, Sigurdsson S, Imsland F, Mauceli E, Engensteiner M, Bailey E, Sandmeyer L, Grahn B, Lindblad-Toh K, Wade CM.Leopard Complex spotting occurs in several breeds of horses and is caused by an incompletely dominant allele (LP). Homozygosity for LP is also associated with congenital stationary night blindness (CSNB) in Appaloosa horses. Previously, LP was mapped to a 6 cm region on ECA1 containing the candidate gene TRPM1 (Transient Receptor Potential Cation Channel, Subfamily M, Member 1) and decreased expression of this gene, measured by qRT-PCR, was identified as the likely cause of both spotting and ocular phenotypes. This study describes investigations for a mutation causing or associated with the Le...
Quan M, Lourens CW, MacLachlan NJ, Gardner IA, Guthrie AJ.Nucleotide sequences of 52 South African isolates of African horse sickness virus (AHSV) collected during 2004-2005 and including viruses of all nine AHSV serotypes, were used to design and develop a duplex real-time reverse transcription quantitative PCR (RT-PCR) assay targeting the VP7 (S8) and NS2 (S9) genes of AHSV. The assay was optimized for detection of AHSV in fresh and frozen blood of naturally infected horses. Assay performance was enhanced using random hexamers rather than gene-specific primers for RT, and with denaturation of double-stranded RNA in the presence of random hexamers. ...
Nielsen SB, Wilhelm K, Vad B, Schleucher J, Morozova-Roche LA, Otzen D.The normal function of equine lysozyme (EL) is the hydrolysis of peptidoglycan residues of bacterial cell walls. EL is closely related to alpha-lactalbumins with respect to sequence and structure and further possesses the calcium binding site of alpha-lactalbumins. Recently, EL multimeric complexes with oleic acids (ELOAs) were shown to possess tinctorial and morphological properties, similar to amyloidal aggregates, and to be cytotoxic. ELOA's interactions with phospholipid membranes appear to be central to its biological action, similar to human alpha-lactalbumin made lethal to tumor cells. ...
Harris SP, Fujiwara N, Mealey RH, Alperin DC, Naka T, Goda R, Hines SA.Immune adult horses have CD8(+) cytotoxic T lymphocytes (CTLs) that recognize and lyse Rhodococcus equi-infected cells in an equine lymphocyte alloantigen (ELA)-A [classical major histocompatibility complex (MHC) class I]-unrestricted fashion. As protein antigens are MHC class I-restricted, the lack of restriction suggests that the bacterial antigens being recognized by the host are not proteins. The goals of this study were to test the hypothesis that these CTLs recognize unique R. equi cell-wall lipids related to mycobacterial lipids. Initial experiments showed that treatment of soluble R. e...
Kitai Y, Kondo T, Konishi E.A complement-dependent cytotoxicity (CDC) assay was established to measure antibodies to the West Nile virus (WNV) nonstructural protein 1 (NS1) in horses. Sera collected from a WNV-infected horse mediated lysis of WNV NS1-expressing cells in a dose-dependent manner at higher percentages than sera from a Japanese encephalitis virus (JEV)-infected horse. The percentages of specific lysis for sera diluted 1:10 to 1:80 were <19.8% (assay cutoff) for almost all of the 100 JEV-infected or uninfected horses tested, in contrast to 55 to 76% in WNV-infected horses. Experimental infection revealed t...
Shelver WL, Thorson JF, Hammer CJ, Smith DJ.Three horses were dosed with dietary zilpaterol and the urine concentrations measured from withdrawal day 0 to withdrawal day 21. The analyses were carried out using both enzyme-linked immunosorbent assay (ELISA) and an ultraperformance liquid chromatography with triple-quadrupole-tandem mass spectrometric detection (UPLC-MS/MS). The UPLC-MS/MS method was developed to provide rapid analysis with positive analyte identification by following three product ions and computing the two independent ion ratios. When urinary zilpaterol concentrations were between 0.2 and 2 ng/mL, the ELISA had interday...
Meehan M, Burke FM, Macken S, Owen P.Streptococcus equi possesses a haem-uptake system homologous to that of Streptococcus pyogenes and Streptococcus zooepidemicus. The system consists of two ligand-binding proteins (Shr and Shp) and proteins (HtsA-C) with homology to an ABC transporter. The haem-uptake system of S. equi differs from that of S. pyogenes and S. zooepidemicus in that Shr is truncated by two-thirds. This study focused on the SeShr, SeShp and SeHtsA proteins of S. equi. Analysis of shr, shp and shphtsA knockout mutants showed that all three proteins were expressed in vitro and that expression was upregulated under co...
Hackett ES, McCue PM.Glucose assessment and regulation are important factors in the treatment of hospitalized horses and foals. Objective: The purpose of this study was to compare glucose measurement by a veterinary glucometer, adjusted by code for use in horses and foals, to a reference chemistry analyzer. It was hypothesized that the veterinary glucometer and reference analyzer would yield similar results and that interpretation of glucose values obtained from a veterinary glucometer would result in clinically appropriate decisions. Methods: Fifty blood samples from adult horses and 50 blood samples from neonata...
Tong T, Bai Y, Liu G, Wang Q, Zhang W, Xiao Y, Xu S, Liu N, Yang T, Wu D.IL-18 is a cytokine originally discovered as an important modulator of immune responses and subsequently shown to be pleiotropic. In this report, we expressed the recombinant equine mature interleukin-18 (rEMIL-18) in E. coli and purified it by nickel affinity gel column chromatography. Purified rEMIL-18 had biological activity commensurate with recombinant human IL-18, as determined by its synergistic effect with recombinant human IL-12 (rhIL-12) on the induction of IFN-gamma gene expression in equine peripheral blood mononuclear cells (PBMC). Following intraperitoneal (i.p.) immunization of ...
Hoogewijs M, Rijsselaere T, De Vliegher S, Vanhaesebrouck E, De Schauwer C, Govaere J, Thys M, Hoflack G, Van Soom A, de Kruif A.Three experiments were conducted to evaluate the impact of centrifugation on cooled and frozen preservation of equine semen. A standard centrifugation protocol (600 x g for 10 min=CP1) was compared to four protocols with increasing g-force and decreased time period (600 x g, 1200 x g, 1800 x g and 2400 x g for 5 min for CP2, 3, 4, and 5, respectively) and to an uncentrifuged negative control. In experiment 1, the influence of the different CPs on sperm loss was evaluated by calculating the total number of sperm cells in 90% of the supernatant. Moreover, the effect on semen quality following ce...
Merwyn S, Kumar S, Agarwal GS, Rai GP.Glanders is highly contagious disease of equines, caused by Burkholderia mallei. The disease though rare, can be transmitted to humans. Here, we report a strategy for rapid detection of B. mallei from environmental samples. Different bacteriological media were evaluated and brain heart infusion broth medium with selective supplements (BHIB-SS) of penicillin (200 U/ml) and crystal violet (1:10,00000) was found to support the maximum growth of B. mallei even in the presence of other bacteria like Escherichia coli and Staphylococcus aureus. A polymerase chain reaction (PCR) and a DNA hybridizatio...
Zhang J, Stein DA, Timoney PJ, Balasuriya UB.A significant consequence of equine arteritis virus (EAV) infection of horses is persistence of the virus in a variable percentage of infected stallions. We recently established an in vitro model of EAV persistence in cell culture for the purpose of furthering our understanding of EAV biology in general and viral persistence in the stallion in particular. In this study we investigated whether persistently infected HeLa cells could be cured of EAV infection by treatment with an antisense peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) designed to target the 5'-terminal region o...
Preziuso S, Laus F, Tejeda AR, Valente C, Cuteri V.Streptococcus (S.) dysgalactiae subsp. equisimilis is responsible for severe diseases in humans, including primary bacteraemia, pneumonia, endocarditis, and toxic shock syndrome. Infection in some animal species can also occur, although a few studies have looked into cross-species infectivity. In horses, S. equisimilis is generally considered infrequent or opportunistic, but has recently been isolated from cases of strangles-like disease. Rapid and sensitive diagnostic techniques could enable epidemiological studies and effective investigation of outbreaks involving these bacteria. In this stu...
Ma G, Lu C, Osterrieder N.A single amino acid variation in the equine herpesvirus type 1 (EHV-1) DNA polymerase (Pol) (D752/N752) determines its neuropathogenic potential. Here, an EHV-1 strain RacL11 mutant with a deletion of Pol residue 752 was constructed. The deletion virus was then repaired to encode D752 or N752, respectively. The Delta752 mutant virus replicated with kinetics indistinguishable from those of D752 and N752 viruses. In addition, we could demonstrate that the deletion mutant was significantly more resistant to aphidicolin, a drug targeting Pol, compared with the N752 but not the D752 variant. In equ...
Jarrett M, Hibbert DB, Osborne R, Young EB.The analysis of total carbon dioxide (TCO(2)) in equine plasma is conventionally done in Australia and elsewhere using Beckman Synchron EL-ISE(R) analysers. This instrument is no longer being manufactured and has not been supported by the supplier since December 2008. For testing for TCO(2) to continue, it is necessary to evaluate and commission alternative instrumentation. In this paper, we compare the Beckman Synchron EL-ISE, the Beckman Synchron CX5, the Beckman UniCel DxC 600 and the Randox Daytona instruments. Results indicate that all four instruments perform in accordance with the manuf...
Grady JG, Elder SH, Ryan PL, Swiderski CE, Rashmir-Raven AM.Hereditary equine regional dermal asthenia (HERDA) is an autosomal recessive skin disorder that has yet to be fully characterized. HERDA is predominately expressed in Quarter Horses, with the majority of these disseminating from elite cutting horse bloodlines, leading to the increased incidence of HERDA in recent years. Affected horses have loose, hyper-extensible, fragile skin and are frequently euthanized due to poor wound healing and disfiguring scars. This study sought to better characterize HERDA by analysis of the biomechanical parameters of tensile strength, modulus of elasticity, energ...
Desantis S, Ventriglia G, Zizza S, Nicassio M, Valentini L, Di Summa A, Lacalandra GM.Stallion sperm from semen collected in Southern Italy during the breeding (June-July) and non-breeding (December-January) periods were analyzed by means of twelve lectins to evaluate the glycoconjugate pattern and to verify whether there are any seasonal differences in the glycosylation pattern of the sperm glycocalyx. The acrosomal cap showed reactivity for Maackia amurensis (MAL II), Sambucus nigra (SNA), Arachis hypogaea (PNA), Glycine max (SBA), Helix pomatia (HPA), Canavalia ensiformis (Con A) Triticum vulgaris (WGA), and Griffonia simplicifolia isolectin II (GSA II) in breeding and non-b...
Tóth F, Frank N, Martin-Jiménez T, Elliott SB, Geor RJ, Boston RC.Hyperinsulinaemia is detected in horses with insulin resistance (IR) and has previously been attributed to increased pancreatic insulin secretion. Connecting peptide (C-peptide) can be measured to assess pancreatic function because it is secreted in equimolar amounts with insulin and does not undergo hepatic clearance. Objective: A human double antibody radioimmunoassay (RIA) detects C-peptide in equine serum and concentrations would reflect responses to different stimuli and conditions. Methods: A validation procedure was performed to assess the RIA. Six mature mares were selected and somatos...
Poirée JC, Vannier C, Sudaka P, Fehlmann M.Brush border membranes isolated from horse kidney cortex as closed right-side out vesicles show selective permeability when analyzed on sucrose and dextran gradients. These vesicles can actively accumulate D-glucose. The preservation of the glucose transport system is demonstrated by the following features: (a) the uptake and release rates of D-glucose are higher in the presence of a sodium gradient, showing that D-glucose transport is a sodium-dependent process; (b) this transport, specific for the D-isomer, is inhibited by phlorizin; (c) the D-glucose transport system is saturable; (d) no in...
Jackson BF, Blumsohn A, Goodship AE, Wilson AM, Price JS.Studies in humans have found circadian changes to be one of the most important sources of controllable preanalytical variability when evaluating bone cell activity using biochemical markers. It remains unclear whether similar circadian changes influence bone marker concentrations in the horse. The aim of this study was to characterize changes in serum concentrations of three biochemical markers of bone cell activity over a 24-h period in six 2-yr-old Thoroughbred mares, and to determine circadian variability in IGF-I, which regulates bone turnover. Three bone markers were measured in serum: os...
Aleman M, Costa LRR, Crowe C, Kass PH.Neuroglycopenia refers to a shortage of glucose in the brain resulting in neuronal dysfunction and death if left untreated. Presumed neuroglycopenia has not been described in horses. Objective: To report neurological signs in horses with presumed neuroglycopenia as the result of severe hypoglycemia. Methods: Ninety horses (hours to 28 years of age) diagnosed with hypoglycemia (blood glucose concentration < 75 mg/dL [< 4.2 mmol/L]). Methods: Retrospective study. Electronic medical records were searched. Signalment, history, complaint, clinical signs, laboratory findings including CSF analysis, ...
Klimas NG, Caldwell KE, Whitney PL, Fletcher MA.Membrane glycoproteins from horse, sheep, goat and bovine erythrocytes were solubilized and purified. These glycoproteins could be placed in three groups based on their degrees of glycosylation: The major bovine erythrocyte glycoprotein (BGII) had 77% sugar, the minor bovine glycoprotein (BGI) had 27% sugar and the others had approximately 50% sugar. Four of the glycoproteins aggregated in a uniform way in aqueous solution--one, BGII, did not. Four had similar subunit sizes of 25-34,000 daltons, but BGII was larger--55,000 daltons. Receptor functions (for plant and invertebrate lectins, antibo...
Nielsen KH.Using antisera specific for the heavy chain of human IgE and bovine reaginic immunoglobulin, the degree of cross-reaction amongst sera from pig, rat, rabbit, guinea pig, goat, cow, horse, dog, cat and human was tested. Antihuman IgE antiserum gave strong reactions with pig, rabbit, cow, goat and human sera (100% to 15.1%) and weak reactions with rat, guinea pig, horse, dog and cat sera (10.1% to 3.22%). Antibovine reagin antiserum produced a considerable amount of cross-reaction with sera from pig, rat, rabbit, goat, horse and human (43.6% to 20.1%) with limited reactions with guinea pig, dog ...
Shen DT, Gorham JR, McGuire TC.An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of equine infectious anemia (EIA) antibody in horse sera. Purified P26 viral protein was the antigen; alkaline phosphatase linked to rabbit anti-horse immunoglobulin G was the conjugate. The ELISA detected EIA antibodies in horse sera as early as 11 to 14 days after experimental inoculations. There was full agreement between the results of ELISA and the agar-gel immunodiffusion tests on EIA proficiency test sera. The ELISA readily detected EIA antibody in horse sera that had weak positive reactions on agar-ge...
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...
Lorenzo PL, Liu IK, Carneiro GF, Conley AJ, Enders AC.Epidermal growth factor (EGF) has been shown to have a positive effect during oocyte in vitro maturation in several species. This study was performed to establish the capacity of equine oocytes to undergo nuclear maturation in the presence of EGF and to localise its receptor in the equine ovary by immunohistochemical methods. Oocytes were obtained by aspiration and subsequent scraping from equine follicles (15-25 mm diameter) and cultured in 3 different treatment groups for 36 h: control Group (modified TCM 199 with 0.003% BSA), EGF Group (TCM-199 supplemented with 50 ng/ml EGF) and EMS Group ...
Choi YH, Landim-Alvarenga FC, Seidel GE, Squires EL.Experiments were conducted to study effects of macromolecules on stallion sperm capacitation and fertilization as determined by penetration of bovine zona-free and equine partially zona-removed oocytes. Stallion sperm were capacitated in TYH medium (modified Krebs-Ringer bicarbonate) supplemented with either 1 mg/mL of polyvinylalcohol (PVA) or 4 mg/mL of BSA. Capacitation was induced with 8 bromoadenosine cyclic monophosphate (8BrcAMP; 0.5 mM) alone or in combination with 0.1 microM of ionomycin. Intraspecies gametes were co-incubated in TYH/PVA or TYH/BSA for 18 to 20 h. For zona-free bovine...
Harvey SP, Minter JM.In this study, the subspecies differentiation of 25 isolates of Burkholderia mallei was attempted based on their ribotype polymorphisms. The isolates were from human and equine infections that occurred at various times around the world. DNA samples from each isolate were digested separately with PstI and EcoRI enzymes and probed with an Escherichia coli-derived 18-mer rDNA sequence to identify diagnostic fragments. Seventeen distinct ribotypes were identified from the combined data obtained with the two restriction enzymes. The results demonstrate the general utility of ribotyping for the subs...
Magnarelli LA, Ijdo JW, Van Andel AE, Wu C, Fikrig E.To develop and evaluate a polyvalent ELISA incorporating a highly specific recombinant antigen (p44) for diagnosis of granulocytic ehrlichiosis in dogs and horses. Methods: 32 dogs and 43 horses. Methods: Results of the ELISA were compared with results of indirect fluorescent antibody (IFA) staining and western immunoblotting incorporating whole-cell antigen. Results: For the canine and equine samples, percentages of samples with positive IFA staining, western immunoblotting, and ELISA results were similar. For 29 (91 %) canine samples and 30 (70%) equine samples, results of IFA staining, west...
Weiland F, Matheka HD, Coggins L, Hatner D.Morphological studies of EIAV reveal knobs on the surface of the particles, conically and tubularly shaped cores, budding particles with dense crescents directly underlying the plasma membrane, and distinct intracytoplasmic structures in infected cells.
Salmon SA, Walker RD, Carleton CL, Robinson BE.A gram-variable pleomorphic bacillus was isolated from the reproductive tracts of 4 mares during routine prebreeding soundness examinations. Using a commercial bacterial identification system, these organisms were identified as Streptococcus acidominimus. However, colonial and Gram-staining characteristics did not support this identification. Subsequent testing indicated the organism was similar to Gardnerella vaginalis. Additional growth and biochemical analysis performed in our laboratory and at the Michigan Department of Public Health and by the Center for Disease Control, Atlanta, Georgia,...
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...
Wagner B.Host immune analyses require specific reagents to identify cellular and soluble components of the immune system. These immune reagents are often species-specific. For horses, various immunological tools have been developed and tested by different initiatives during the past decades. This article summarizes the development of well characterized monoclonal antibodies (mAbs) for equine immune cells, immunoglobulin isotypes, cytokines, and chemokines.
Wang CC, Hartmann-Fischbach P, Krueger TR, Wells TL, Feineman AR, Compton JC.3,4-Methylenedioxypyrovalerone (MDPV) is a psychoactive drug with potent stimulant properties and potential for abuse and drug dependency. MDPV was recently classified as a Class I drug by Racing Commissioners International, indicating that it is a banned substance in equine athletes because it lacks therapeutic value in horses. To enforce this ban, a sensitive and fast liquid chromatography-tandem mass spectrometry method was needed. It is for this reason that this method was developed for quantification and confirmation of MDPV in equine plasma. Sample preparation involved liquid-liquid extr...
Wang Y, Mao Q, Chang H, Wu Y, Pan S, Li Y, Zhang Y.Integrins can function as receptors for foot-and-mouth disease virus (FMDV) in epithelium. Horses are believed to be insusceptible to this disease, but the mechanism of resistance remains unclear. To detect whether FMDV can use integrin to attach to equine epithelial, we compared the utilities of αvβ3 and αvβ6 between bovine and equine kidney epithelial cells (KECs). Equine KECs showed almost equal efficiency to those of bovine. Further, the integrin αv, β3, and β6 subunits from bovine and equine were cloned and vectors were transfected into SW480 cells and COS-1 cells alone or together...
Szczerba-Turek A, Siemionek J, Ras A, Bancerz-Kisiel A, Platt-Samoraj A, Lipczynska-Ilczuk K, Szweda W.Equine sarcoids are the most common neoplasms in horses. Bovine papilloma- virus type 1 (BPV-1) is the main viral type identified in equine sarcoids in Europe. Objective: The aim of the present study was to genetically evaluate BPV types based on DNA analyses of the CDS of the L1 gene. The presence of BPV DNA was confirmed by Degenerate Oligonucleotide-Primed Polymerase Chain Reaction (DOP PCR) with FAP59/FAP64 consensus primers. Results: The DNA was detected in 21/40 (52.5%) of clinically diagnosed sarcoids. More than half of 14 isolates (66.7%) shared 100% homology with BPV-1 Deltapapillomav...
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. ...
Pawlak EA, Geor RJ, Watts MR, Black SJ, Johnson PJ, Belknap JK.Hypoxia-inducible factor-1α (HIF-1A) is an important protein in the regulation/induction of many genes in the cellular and tissue response to hypoxia and a central mediator in inflammatory signalling. As both hypoxia and inflammatory events are purported to occur in the lamellar epidermis in sepsis-related laminitis in the equid, HIF-1A may play a central role in this disease process. Objective: To assess the regulation of HIF-1A and HIF-1A-related genes in the equine keratinocyte in vitro and in the lamellar tissue of horses with sepsis-related laminitis. Methods: In vivo and in vitro experi...
Anderson ME, Weese JS.Screening for nasal colonization is an important aspect of many methicillin-resistant Staphylococcus aureus (MRSA) control programs. Real-time polymerase chain reaction (RT-PCR) is an attractive alternative to standard culture techniques because of the considerably shorter turnaround time. An assay has been validated for diagnostic purposes in humans, however this methodology has not been evaluated in horses. The purpose of this study was to compare an RT-PCR assay for rapid identification of MRSA directly from nasal swabs in horses to standard culture techniques. Nasal swabs collected from 29...
Sabir N, Chaudhry ZI, Aslam A, Muhammad K, Shahid M, Hussain A, Khan SA, Ahmad I.Trypanosomiasis is an important protozoal disease with a diverse range of susceptible host including human. In the current study, molecular characterization of prevalent species was done through a pan-trypanosome polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP). A total of three hundred (n = 300) equines including horses, donkeys and mules (100 each) were randomly selected and the equine blood samples were subjected to screening for trypanosomes through microhaematocrit centrifuge technique (MHCT), conventional PCR, semi-nested PCR and RFLP. Overall...
Dunkel B, Bolt DM, Smith RK, Cunningham FM.Platelet-rich plasma (PRP) is increasingly used for treatment of orthopaedic injuries. However, the effects of different stimuli on the release pattern of regenerative and proinflammatory factors from equine platelets are largely unknown and an optimal treatment protocol remains to be established. Objective: The aim of this study was to identify a stimulus that enhanced release of histopromotive factors (platelet-derived growth factor BB [PDGF] and transforming growth factor 1β[TGF]) without causing concurrent release of a proinflammatory mediator (CCL5). Methods: Washed platelets were prepar...
Kulmala KA, Pulkkinen HJ, Rieppo L, Tiitu V, Kiviranta I, Brünott A, Brommer H, van Weeren R, Brama PA, Mikkola MT, Korhonen RK, Jurvelin JS....Contrast-enhanced computed tomography (CECT) has been introduced for the evaluation of cartilage integrity. Furthermore, CECT enables imaging of the structure and density of subchondral bone. In this laboratory study, we investigate the potential of microCECT to simultaneously image cartilage and subchondral bone for the evaluation of tissue healing. Methods: Osteochondral lesions (Ø = 6 mm) were surgically created in equine intercarpal joints (n = 7). After spontaneous healing for 12 months, the horses were sacrificed and osteochondral plugs (Ø = 14 mm), including the repair cartilage and a...
Satterlee JD, Moench S.Proton hyperfine resonance assignments for cytochromes c from several species are currently being successfully pursued by several laboratories. These efforts focus mostly on the ferrous forms. In contrast to that work, we have pursued assignments of the proton hyperfine shifted resonances for horse and tuna ferricytochromes c. Our results indicate that assignments are nearly identical in those two proteins. Using the pre-steady state nuclear Overhauser effect, several additional assignments have been made for the tuna protein, whereas for the horse protein, the following protons have been assi...
Davis RO, Gravance CG, Casey PJ.Tissue variation in microscope slides made for spermatozoon analysis and variation introduced by the subjective techniques used to analyze these slides reduce the statistical power of studies that seek to use spermatozoon morphology to predict fertility. A simple specimen preparation method was developed to standardize stallion spermatozoon morphologic smears, and a new, automated spermatozoa morphometry instrument was used to objectively analyze the efficacy of the specimen preparation technique. The method achieved a standard spermatozoon concentration and reduced field-to-field variation in...
Main AR, Soucie WG, Buxton IL, Arinc E.A relatively simple method is described by which cholinesterase was purified about 19000-fold starting from horse serum. Typically 20 litres of serum were processed to yield 15-18mg of electrophoretically pure cholinesterase in the form of an active salt-free dry powder. The method included two stages: fractionation with (NH(4))(2)SO(4) and ion-exchange chromatography. The (NH(4))(2)SO(4) stage included, in principle, the acid (pH3) step of the Strelitz (1944) procedure. The step took advantage of the stabilizing effect that 33%-satd. (NH(4))(2)SO(4) has on cholinesterase activity at pH3 and i...
Ishii M, Kobayashi S, Acosta TJ, Miki W, Yamanoi T, Matsui M, Miyake Y, Miyamoto A.The aim of this study was to clarify the relationship between circulating oxytocin (OT) and PGF(2alpha) metabolite (PGFM) in mares at the third stage of labor and placental expulsion time in order to investigate a cause of retained placenta of which the incidence increase in a heavy draft mare. Blood was sampled every 5 min from foaling to expulsion of the placenta in 18 heavy draft mares to evaluate circulating OT and PGFM. The relationships between OT and PGFM concentration and recorded placental expulsion times were investigated. The results were as follows (1) The highest level of OT conce...
Gerst S, Borchers K, Gower SM, Smith KC.EHV-1 and EHV-4 abortion diagnosis is based upon detailed examination of the aborted fetus. However, in some cases, only the placenta is available for examination. Furthermore, the contribution of lesions in the placenta to pathogenesis and diagnosis of EHV-1 and EHV-4 abortion has been neglected. Objective: To assess the utility of placental examination in equine herpesvirus-1 (EHV-1) and EHV-4 abortion diagnosis. Methods: Sections of allantochorion from 49 herpesvirus abortions were analysed by PCR, in situ hybridisation and immunostaining. Results: Virus-specific nested PCR confirmed the pr...
Cox KS, Nelson BB, Wittenburg L, Gold JR.Regional limb perfusion (RLP) is an effective treatment option for injuries and infections of the distal limb in horses. Using ceftiofur sodium in RLP has been studied due to its superior spectrum of Gram-positive organisms compared to aminoglycosides, but it is not known if this antimicrobial drug adequately penetrates subcutaneous tissue and bone. Objective: To determine the concentration of ceftiofur in plasma, subcutaneous tissue and bone in horses after RLP. Methods: Experimental prospective study. Methods: Six healthy horses were used in this study. Under standing sedation, an Esmarch to...
