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.
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...
Nemoto M, Imagawa H, Tsujimura K, Yamanaka T, Kondo T, Matsumura T.Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was applied to detection of equine rotavirus. Because equine rotavirus of the single P genotype, P[12], is predominant in the equine population worldwide, an RT-LAMP primer set was designed to target the genotype P[12] sequence and thus detect equine rotavirus. The detection limit of the RT-LAMP assay was 10(3) copies of viral RNA, whereas that of semi-nested RT-PCR for genotype P[12] was 10(5) copies. The RT-LAMP assay specifically amplified genotype P[12] but did not amplify the other P genotype strains. The RT-LAMP assay...
Buss DG, Giuliano EA, Sharma A, Mohan RR.To establish an in vitro model for the investigation of equine corneal wound healing. To accomplish this goal, a protocol to isolate and culture equine corneal keratocytes, fibroblasts and myofibroblasts was developed. ANIMAL MATERIAL: Equine corneal buttons were aseptically harvested from healthy research horses undergoing humane euthanasia for reasons unrelated to this study. Slit-lamp biomicroscopy was performed prior to euthanasia by a board-certified veterinary ophthalmologist to ensure that all samples were harvested from horses free of anterior segment disease. Methods: Equine corneal s...
Yu NH, Ho EN, Wan TS, Wong AS.Recombinant human erythropoietin (rhEPO), darbepoetin alfa (DPO) and methoxy polyethylene glycol-epoetin beta (PEG-EPO) are synthetic analogues of the endogenous hormone erythropoietin (EPO). These erythropoiesis-stimulating agents have the ability to stimulate the production of red blood cells and are commercially available for the treatment of anaemia in humans. These drugs are understood to have performance-enhancing effects on human athletes due to their stimulation of red blood cell production, thereby improving delivery of oxygen to the muscle tissues. Although their effect on horses has...
Mancini RA, Suman SP, Konda MK, Ramanathan R, Joseph P, Beach CM.Research focused on determining the fundamental mechanisms by which lactate influences color stability has not considered a direct effect of lactate on myoglobin. Thus, the objective of this study was to use Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry to examine lactate adduction to myoglobin. Equine oxymyoglobin and equine carboxymyoglobin (0.15mM) were incubated with sodium lactate (200mM) at 4 degrees C, pH 5.6 in 50mM sodium citrate buffer or at 37 degrees C, pH 7.4 in 50mM sodium phosphate buffer, simulating typical meat storage and physiological condition...
Chung TH, Park GB, Lim CY, Park HM, Choi GC, Youn HY, Chae JS, Hwang CY.Identification of the species and strain of dermatophyte can play an effective role in control of disease outbreaks by establishing the source of infection. Current methods of identification are based on cultural and microscopic methods, often involving weeks before a positive identification are made. A rapid molecular diagnostic method would therefore be an important laboratory technique, but requires confirmation in equine clinical practice. Objective: To test the sensitivity and specificity of molecular diagnostic methods applied to a racehorse herd from the Korean Racehorse Authority (KRA)...
Hillström A, Tvedten H, Lilliehöök I.An in-clinic assay for equine serum amyloid A (SAA) analysis, Equinostic EVA1, was evaluated for use in a clinical setting. Stability of SAA in serum samples was determined. Methods: Intra- and inter- assay variation of the in-clinic method was determined. The in-clinic method (EVA1) results were compared to a reference method (Eiken LZ SAA) with 62 patient samples. For samples with SAA concentrations within the assay range of EVA1 (10-270 mg/L), differences between the methods were evaluated in a difference plot. Linearity under dilution was evaluated in two samples. Stability of SAA in three...
Ascenzi P, di Masi A, Gullotta F, Mattu M, Ciaccio C, Coletta M.Cyanide binds to ferric heme-proteins with a very high affinity, reflecting the very low dissociation rate constant (k(off)). Since no techniques are available to estimate k(off), we report herewith a method to determine k(off) based on the irreversible reductive nitrosylation reaction to trap ferric myoglobin (Mb(III)). The k(off) value for cyanide dissociation from ferric cyanide horse heart myoglobin (Mb(III)-cyanide) was determined at pH 9.2 and 20.0 degrees C. Mixing Mb(III)-cyanide and NO solutions brings about absorption spectral changes reflecting the disappearance of Mb(III)-cyanide w...
Gosálvez J, Crespo F, Vega-Pla JL, López-Fernández C, Cortés-Gutiérrez EI, Devila-Rodriguez MI, Mezzanotte R.The genome of stallion (Spanish breed) and donkey (Spanish endemic Zamorano-Leonés) were compared using whole comparative genomic in situ hybridization (W-CGH) technique, with special reference to the variability observed in the Y chromosome. Results show that these diverging genomes still share some highly repetitive DNA families localized in pericentromeric regions and, in the particular case of the Y chromosome, a sub-family of highly repeated DNA sequences, greatly expanded in the donkey genome, accounts for a large part of the chromatin in the stallion Y chromosome.
Byrne BA, Rand CL, McElliott VR, Samitz EM, Brault SA.Dermatophilosis caused by Dermatophilus congolensis causes exudative dermatitis in a variety of species. The infection is generally limited to the cutaneous tissues, where infection is acquired from the environment and bacteria penetrate into keratinized epithelium through epithelial disruption. A 3-year-old pony filly was examined for enlarging mandibular lymph nodes during the preceding 10 months. Biopsy of the node revealed mixed and granulomatous inflammation and thick, filamentous Gram-positive bacteria. Dermatophilus congolensis, confirmed by biochemical testing and sequencing of the rib...
Nemoto M, Tsujimura K, Yamanaka T, Kondo T, Matsumura T.Loop-mediated isothermal amplification (LAMP) is a novel method for the rapid and sensitive detection of DNA without the need for expensive equipment. In the present study, LAMP assays were developed for the specific detection of Equid herpesvirus 1 and 4 (EHV-1 and EHV-4, respectively) and for the differentiation of glycoprotein E (gE)-deleted EHV-1 (DeltagE) strain, a candidate strain for a live vaccine, from field EHV-1 strains. Specific primer sets were designed for the gC and gE genes of EHV-1 and for the gC gene of EHV-4. The analytical sensitivities of the LAMP assays were compared with...
Zullian C, Menozzi A, Pozzoli C, Poli E, Bertini S.The effects of selective α(2)-agonists (xylazine, detomidine and medetomidine) and antagonists (yohimbine and atipamezole) on in vitro small intestine motility in the horse were evaluated. Samples of equine jejunum were placed in isolated organ baths and drug-induced modifications of motility were measured by means of an isotonic transducer. All tested α(2)-agonists dose-dependently reduced both spontaneous and electrically-evoked phasic contractions. Conversely, α(2)-antagonists were ineffective when tested alone, and showed a heterogeneous and dose-independent ability to inhibit agonist a...
Meyer W, Hornickel I, Schoennagel B.Using the zinc-iodide osmium tetroxide (ZIO) method, TEM and immunohistochemistry (for CD1a and langerin), the study demonstrates Langerhans cells in the oesophageal epithelium of domesticated mammals (herbivores: horse, cattle, goat; omnivores: pig, dog, laboratory rat; carnivores: cat), although with variations between the species. The ZIO method and TEM showed this cell type in the cat and, sporadically, in the horse; CD1a (+) Langerhans cells were demonstrated in the ovine, porcine and murine oesophagus. Positive staining for langerin was detected in single cells of the caprine, canine, mu...
Patterson SD, Bell K.Three cases of abnormal expression of the equine protease inhibitory alleles, Pi F, L, and S1, were observed following the examination of 30,000 plasma samples by one-dimensional acid (pH 4.6) polyacrylamide gel electrophoresis. Characterization of the abnormal proteins in terms of isoelectric point, molecular mass, inhibitory spectra, and sialic acid content was performed using one- and two-dimensional electrophoretic techniques. The Pi F and S1 abnormalities were postulated to be the result of amino acid substitutions causing alterations in the processing of the carbohydrate side chains. No ...
Paris DB, Kuijk EW, Roelen BA, Stout TA.Real-time quantitative PCR (qPCR) is invaluable for investigating changes in gene expression during early development, since it can be performed on the limited quantities of mRNA contained in individual embryos. However, the reliability of this method depends on the use of validated stably expressed reference genes for accurate data normalisation. The aim of the present study was to identify and validate a set of reference genes suitable for studying gene expression during equine embryo development. The stable expression of four carefully selected reference genes and one developmentally regula...
Conti A, Godovac-Zimmermann J, Liberatori J, Braunitzer G.beta-Lactoglobulin-like proteins were detected in horse colostrum and normal milk using immunological techniques. In contrast to the beta-lactoglobulins sequenced so far these proteins are monomeric and genetically not homogenous. In this paper we report the first primary structure of a monomeric beta-lactoglobulin from horse colostrum. By means of an automatic liquid-phase sequenator the sequence of peptides obtained by tryptic digestion and by cyanogen bromide cleavage was determined. A limited tryptic digestion and hydrolysis with chymotrypsin provided the necessary overlapping peptides. Th...
Chen JW, Uboh CE, Soma LR, Li X, Guan F, You Y, Liu Y.Proper identification of racehorses competing in an official race and maintenance of defensible chain of custody are important in doping control regulations. The purpose of this study was to develop a reliable multiplex PCR method for providing genetic evidence for matching donors to test samples by using short tandem repeat (STR) loci. Amplification of 21 STR loci from blood, urine or hair root was achieved in a single tube and STR length polymorphism was analyzed using fluorescent labeled capillary electrophoresis. This novel approach showed an allele confidence interval of 0.19-0.43 bp and ...
Burns SJ, Simpson RB, Snell JR.Sterile equipment was used to collect ten ejaculates from each of ten normal stallions and quantitative and qualitative bacterial counts were made within 15 min after collection. The mean bacterial population in undiluted semen was found to be 573,000 +/- 374,000 organisms/ml. The bacterial content of semen diluted with two parts sterile physiological saline, or with two parts of a cream-gelatin extender, was measured within 15 min after collection and again after 2 hr at 25 degrees C. The number of bacteria was slightly increased in the saline after 2 hr, but 1 93% and 99% reduction occurred ...
McDowell KJ, Sharp DC, Fazleabas AT, Roberts RM.Conceptuses were obtained from pony mares on each day of pregnancy between Days 12 and 28, and on Days 39, 45, 65 and 100. Endometrium was obtained from mares at Days 12, 14, 16, 18, 39, 45, 65 and 100 of pregnancy, and from non-pregnant mares during anoestrus, during transition into the breeding season, at oestrus, or during dioestrus. Tissues were incubated in vitro for 24 h with L-[3H]leucine. Proteins synthesized and released into the culture medium were analysed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and fluorography. Conceptuses obtained before Day 14 after ovul...
Pusterla N, Pusterla JB, Spier SJ, Puget B, Watson JL.The SNAP Foal IgG test (IDEXX) as evaluated for its accuracy and usefulness by measuring blood samples collected from 42 foals between 24 and 48 hours after birth. The results were compared with the single radial immunodiffusion (SRID) test as the reference method. The SNAP test was quick and easy to perform, and the results were similar to those obtained by SRID in 64 per cent of the samples. The best results were found with low ( 800 mg/dl) concentrations of immunoglobulin G, with an accuracy of 80 per cent and 89 per cent, respectively. The intermediate concentrations were usually lower whe...
Pisano MB, Seco MP, Ré VE, Farías AA, Contigiani MS, Tenorio A.Venezuelan Equine Encephalitis (VEE) complex belongs to alphavirus genus in the family Togaviridae. Several species of this complex are pathogenic to humans. VEE infections can produce severe or mild disease, and many cases remain undiagnosed. A specific and sensitive reverse transcriptase nested polymerase chain reaction (RT-Nested PCR) method was developed for the detection of all VEE subtypes, including Rio Negro Virus (RNV) (subtype VI), which circulates only in Argentina. Degenerated primers were designed and thermal cycling parameters were standardized. This technique is suitable for rap...
Vidament M, Magistrini M, Le Foll Y, Levillain N, Yvon JM, Duchamp G, Blesbois E.This study tested whether variable temperatures (from -0.5 to 15 °C) and air exposure could be used under laboratory and under field conditions to store stallion sperm diluted in extender INRA96 without loss of fertility. Experiment 1 (laboratory conditions) measured the effects of two 72 h storage conditions (5 °C with air vs. 15 °C without air). Experiment 2 (fixed field conditions) measured the effects of 22 h of storage without air in disposable containers maintained at four ambient temperatures (7 °C, 17 °C, 27 °C, 39 °C with semen at -0.5 °C to 3 °C, 4 °C to 7 °C, 8 °C to 10 ...
Hirsbrunner G, Reist M, Couto SS, Steiner A, Snyder J, Vanleeuwen E, Liu I.Uterine smooth muscle specimens were collected from euthanatized mares in estrus and diestrus. Longitudinal and circular specimens were mounted in organ baths and the signals transcribed to a Grass polygraph. After equilibration time and 2 g preload, their physiologic isometric contractility was recorded for a continuous 2.0 h. Area under the curve, frequency and time occupied by contractions were studied. Differences between cycle phases, between muscle layers, and over the recorded time periods were statistically evaluated using linear mixed-effect models. In the mare, physiologic contractil...
Balkema-Buschmann A, Fischer K, McNabb L, Diederich S, Singanallur NB, Ziegler U, Keil GM, Kirkland PD, Penning M, Sadeghi B, Marsh G, Barr J....Since the identification of Hendra virus (HeV) infections in horses in Australia in 1994, more than 80 outbreaks in horses have been reported, and four out of seven spillover infections in humans had a fatal outcome. With the availability of a subunit vaccine based on the HeV-Glycoprotein (HeV-G), there is a need to serologically ifferentiate the nfected from the accinated nimals (DIVA). We developed an indirect ELISA using HeV-G expressed in and HeV-Nucleoprotein (HeV-N) expressed in recombinant baculovirus-infected insect cells as antigens. During evaluation, we tested panels of sera from n...
Janczyk P, Weigner J, Luebke-Becker A, Richardson KC, Plendl J.Most embalming of cadavers for anatomical dissection in veterinary medicine has used 6-10% formaldehyde resulting in discoloured and rigid specimens. This project produced teaching specimens of sheep, horse and calf cadavers having their musculoskeletal and visceral structures with a natural appearance using a fixation solution with lowered concentrations of formaldehyde (2% and 3%) together with ethanol and polyethylene glycols. Fixation parameters (palpable consistency, flexibility, colour, tissue hydration and odour) were assessed qualitatively by twice weekly dissections over two months fo...
Kawai S, Igarashi I, Abgaandorjiin A, Ikadai H, Omata Y, Saito A, Nagasawa H, Toyoda Y, Suzuki N, Matsuda H.In-vitro-propagated Babesia caballi parasites were examined by scanning and transmission electron microscopy. Many small pores were observed over the entire surface of infected erythrocytes on scanning electron microscopy, and on transmission electron microscopy these small pores were found to be openings of tubular structures. By the examination of a number of infected cells the tubular structures were found to be connected with the parasite, and this observation might indicate that the tubular structures arose the edge of the parasite and terminated at an Invagination on the surface of the e...
Cruz A, Sullivan DB, Doty KF, Hess RA, Canisso IF, Reddi PP.The acrosome plays a critical role in sperm-oocyte interactions during fertilization. SP-10 is an acrosomal matrix protein, which is evolutionarily conserved among mammals. The SP-10 antibody has been shown to be useful for staging the seminiferous cycle in the mouse and human. A canonical acrosomal marker; however, has never been used for staging in the horse. The objectives of the present study were to investigate the presence of SP-10 within the horse acrosome using an anti-mouse SP-10 antibody, to classify spermatids based on the shape of the acrosome, and then to use that information to a...
Vivrette SL, Sellon DC, Gibbons DS.Diagnosis of pneumonia caused by Rhodococcus equi can be made more rapidly by use of a polymerase chain reaction (PCR) assay than by use of conventional bacteriologic culture techniques. Use of a PCR assay aids in the differentiation between virulent and avirulent strains of R equi, and the assay may be used to identify R equi in feces and soil of breeding farms.
Thorsteinsdóttir L, Torfason EG, Torsteinsdóttir S, Svansson V.Horses are hosts to 2 types of gammaherpesviruses, Equid herpesvirus 2 and 5 (EHV-2 and EHV-5, respectively). Both EHV-2 and EHV-5 are common in horses in Iceland. An Icelandic EHV-5 isolate was recovered by sequential culture in primary fetal horse kidney and rabbit kidney cells. Glycoprotein B, glycoprotein H, and DNA terminase genes of the isolate were fully sequenced, and the DNA polymerase gene was partly sequenced. To date, the glycoprotein B gene of EHV-5 was the only gene that has been reported to be completely sequenced in addition to small parts of the glycoprotein H, DNA polymerase,...
Aalsburg AM, Erdman MM.Taylorella equigenitalis is the etiologic agent of contagious equine metritis (CEM), a venereal disease of horses. A total of 82 strains of T. equigenitalis isolated in the United States were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion of genomic DNA with restriction enzyme ApaI. Twenty-eight of those strains isolated from horses in the 2009 U.S. outbreak (CEM09) were further analyzed with NotI and NaeI enzymes. When ApaI alone was used for analysis, the 82 isolates clustered into 15 different genotypes that clearly defined groups of horses with known epidemiological co...
Lin G, Chen GH, Ho HC.Conformationally restricted carbamate inhibitors, exo-2-norbornyl-N-butylcarbamate (1), endo-2-norbornyl-N-butylcarbamate (2), l-adamantyl-N-butylcarbamate (3), and 2-adamantyl-N-butylcarbamate (4) as active site-directed irreversible inhibitors of horse serum butyrylcholinesterase are investigated for values of the dissociation constant (KI), the carbamylation constant (k2), and the bimolecular rate constant (ki). Compound 1 is the most potent inhibitor of the enzyme and the values of KI and ki are 20 nM and 1.1 x 10(5) M-1sec-1, respectively.
Ferrante A, Thong YH.Results presented show that highly purified populations of mononuclear (MN) and polymorphonuclear (PMN) leucocytes can be obtained from horse blood by a procedure similar to that previously described for the separation of these leucocytes from human blood. This involved centrifugation of horse blood on a Ficoll-Hypaque medium with a density of 1.095 g/ml. The procedure required approximately 1 h for completion and resulted in the simultaneous preparation of MN (greater than 98% purity) and PMN (greater than 96% purity) leucocytes. Cell viability exceeded 95% and cells retained immunological fu...
Smith HT, Millett F.Spectrophotometric titrations of five singly modified horse heart ferricytochromes c, specifically (trifluoromethyl)phenylcarbamylated (CF3PhNHCO-) or trifluoroacetylated (CF3CO-) at lysines-13, -72, and -79, were carried out. The CF3PhNHCO-Lys-13, Lys-79, and CF3CO-Lys-79 derivatives all underwent alkaline isomerization with loss of the 695-nm band to low-spin species with an apparent pK of about 8.9, as did the unmodified cytochrome. However, modification of lysine-72 appeared to alter the reaction pathway since the CF3PhNHCO-Lys-72 derivative isomerized to a high-spin form with an apparent ...
Washburn LR, Somerson NL.Crude lipoprotein-containing fractions obtained from sera of three different animal species were tested, in combination with bovine serum in Mycoplasma pneumoniae culture medium. All sera yielded at least one lipoprotein-containing component which was considerably more effective in promoting mycoplasma growth than the unfractionated serum sample from which it was derived. The very low activity of certain whole-serum samples tested in this investigation suggests that toxic substances may be present in whole serum which are not contained in the lipoprotein preparations. The greatest activity app...
Bonometti S, Menarim BC, Reinholt BM, Ealy AD, Johnson SE. To provide insight into maternal recognition of pregnancy control in equids, the mitogenic and developmental effects of endometrium-expressed growth factors (epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1)) were examined in equine iTr cells, an equine trophectoderm cell line. Initial western blots revealed that HGF and IGF-1 stimulate phosphorylation of AKT serine/threonine kinase 1 (AKT1) and EGF, FGF2, or HGF resulted in phosphorylation of both extracellular signal-regulated kinase 1 (ERK1) and ERK2...
Larghi OP, González E, Held JR.The corneal test (CT) for rabies diagnosis was evaluated in samples from 313 subjects of different species. Some of the subjects were inoculated experimentally and others were naturally infected. When the CT was compared with immunofluorescence staining and mouse inoculation tests on brains of the same subjects, a sensitivity of 41.7% and a specificity of 100% were found. The authors conclude that a positive CT result would confirm the diagnosis of rabies, but a negative one would not exclude the possibility of disease.
Lo Giudice R, Rizzo G, Centofanti A, Favaloro A, Rizzo D, Cervino G, Squeri R, Costa BG, La Fauci V, Lo Giudice G.The use of equine bone blocks is widely reported for bone augmentation techniques. The block must be shaped according to the form of the defect that should be regenerated. The shaping could be performed by hand before or during the surgery, in a sterile ambient, or using a CNC milling machine that could not be sterile. The aim of our study was to evaluate if a steam sterilization could provide a medical grade sterilization of the blocks and to evaluate if bone microstructure and collagen structures change after different steam sterilization protocols provided by mainstream autoclave. Methods: ...
Fitzgerald TA, Browning GF.The sensitivity of a rotavirus serotyping enzyme-linked immunosorbent assay (ELISA) was improved by the addition of 0.5 mM CaCl2 to the washing buffer and reagent diluent. Twenty-nine of 63 (46%) previously untyped bovine and equine faecal rotavirus samples were serotyped in the modified assay. A differential response to Ca2+ ions was noted for different G-serotypes suggesting that serotyping assays performed without the inclusion of CaCl2 in the assay buffers may produce biased results.
Wuri L, Agca C, Agca Y.This study compared the morphometric, subcellular characteristics, in vitro fertilisation (IVF) and embryonic developmental potential of metaphase II (MII) mouse oocytes obtained from females superovulated with either anti-inhibin serum-human chorionic gonadotrophin (AIS-hCG) or pregnant mare serum gonadotrophin (PMSG)-hCG. The oocyte's quantity, quality, zona pellucida (ZP) thickness, perivitelline space (PVS), diameter, microtubules, F-actin, cortical granules (CGs) and mitochondrial distribution were determined. Superovulation using AIS-hCG resulted in a higher numbers of oocyte/donor compa...
Davis JL, Kirk LM, Davidson GS, Papich MG.To determine the effects of temperature and light over a 35-day period on stability of pergolide mesylate after compounding in an aqueous vehicle. Methods: Evaluation study. Methods: Pergolide was compounded into a formulation with a final target concentration of 1 mg/mL. Aliquots of the formulation were then stored at -20 degrees, 8 degrees, 25 degrees, or 37 degrees C without exposure to light or at 25 degrees C with exposure to light for 35 days. Samples were assayed in triplicate by means of high-pressure liquid chromatography immediately after compounding and after 1, 7, 14, 21, and 35 da...
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...
