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.
Shaktivesh , Malekipour F, Whitton C, Lee PVS.Standard fatigue testing of bone uses a single load and frequency applied until failure. However, in situ, the subchondral bone of Thoroughbred racehorses is subjected to a combination (or a spectrum) of loads and frequencies during training and racing. Objective: To investigate the use of a fatigue testing method for equine third metacarpal (McIII) subchondral bone under a spectrum of loading conditions which a racehorse is likely to experience during a fast workout. Methods: In vitro biomechanical experimental study. Methods: McIII subchondral bone specimens (n = 12) of racehorses were ha...
Ripley AM, Penedo MCT, Grahn RA, Martinez de Andino EV, Walbornn SR, Serafini R, Love CC, Hinrichs K.Fetal genotyping has important applications in the horse, but currently necessitates embryo recovery and biopsy. We investigated whether fetal genotyping could be performed on yolk-sac fluid recovered from pregnant mares via transvaginal aspiration. Fluid was collected before Day 30 to provide results before establishment of the endometrial cups (Day 37). Genotyping and assessment of maternal DNA contamination was performed by analyzing histograms of PCR results for 19 loci. In Exp. 1, mares underwent yolk-sac aspiration on Days 22-28 of gestation. Fluid (0.56-1.02 mL) was recovered from fiv...
Dang W, Shang S, Zhang X, Yu Y, Irwin DM, Wang Z, Zhang S.Previous studies investigating donkey parentage and genetic diversity used horse-specific multiplex systems. However, several mis-allele and null-allele issues were found with some of the horse primers when used in donkeys. In 2017, the International Society for Animal Genetics (ISAG) recommended 13 dinucleotide short tandem repeats (STRs) (AHT4, ASB23, HMS2, HMS3, HMS6, HMS7, HMS18, HTG7, HTG10, TKY297, TKY312, TKY337 and TKY343) as a core panel that should be used to identify individuals and to test for parentage in donkeys. To date, no single multiplex STR typing system containing all 13 do...
Lechmann J, Schoster A, Ernstberger M, Fouché N, Fraefel C, Bachofen C.Equid alphaherpesvirus 1 (EHV-1) infections can have a major impact on the horse industry and equine welfare by causing abortion or respiratory or neurologic disease. A single nucleotide polymorphism (A→G) in open reading frame (ORF) 30, encoding the catalytic subunit of the DNA polymerase, has been shown to be a strong predictive marker for neuropathogenicity. Given that a previously established real-time PCR (rtPCR) protocol yielded unsatisfactory results concerning determination of the EHV-1 genotype, we developed and evaluated a new conventional PCR protocol enabling identification of th...
Farah Haziqah MT, Nur Hikmah AM, Mat Hasan H, Hamdan A, Nik Him NAII.An investigation was undertaken for screening and isolating nematophagous-fungi from the faecal samples of various grazing animals and soils in Malaysia. Total of 111 faeces and 50 soil samples were collected and the samples were cultured on 2% water agar plates. The growth of nematophagous-fungi was stimulated by sprinkling-baiting technique. The conidia of suspected nematophagous-fungi were inoculated on 2% water agar plates. All isolated were maintained on 2% cornmeal agar plates. Verticillium spp., Fusarium spp. and Arthrobotrys spp. were identified from the faecal and soil samples. 62.5% ...
Denham J, Stevenson K, Denham MM.Telomeres are genetically conserved repetitive terminal DNA that protect against genomic instability and shorten with ageing. Here, we reveal the leukocyte telomere length of Equus caballus by measuring terminal restriction fragments (TRFs) using Southern Blot analysis in a cohort of 43 Thoroughbred horses (age: 24 h-25 years). Heterogeneous TRFs were observed in each animal and large inter-animal variation in mean TRF was observed (range: 10.5-18.7 kbp). Mean TRFs were inversely correlated with age (r = -0.47). The estimated yearly rate of telomere attrition was 134 bp. Horses shoul...
Forsyth J, Halliwell RE, Harrand R.Identification of environmental allergens in horses with allergic disease facilitates allergen avoidance and targeted immunotherapy. Objective: To evaluate allergenic co-reactivity between 44 environmental allergens. Methods: Horses with suspected allergic disease (n = 344) whose sera were submitted for environmental allergen testing. Methods: Allergen-specific IgE serological assays were performed using 44 allergens divided into six taxonomically related groups: house dust/storage mites, moulds, insects, grass, tree and weed pollens. Using pairwise comparisons, odds ratios (ORs) were calcul...
Rendle DI, Armstrong SK, Heller J, Hughes KJ.Point-of-care glucometry is used commonly in clinical and research settings; however, accuracy and precision of this method are concerns. The objectives of this study were to determine the accuracy of glucometry in adult horses and the precision of duplicate measurements. Blood samples were collected from 62 horses into one plain syringe, one EDTA tube and three fluoride oxalate (FO) tubes. Immediately after collection, glucose concentrations in whole blood were determined, in duplicate, by glucometry from the syringe (plain whole blood [WB] group), EDTA tube (EDTA group) and one FO tube (FO g...
Adam CD.The formation and release under gravity of blood droplets from simulated weapon tips has been investigated experimentally and the results analysed and interpreted using established theoretical models for detached pendent drops. Droplets were produced from a series of conical nozzles, manufactured with a range of cone angles and including a set of un-bored conical tips, under conditions where the tip was either non-wetted or pre-wetted with blood. For the former, radius-limited case, detached droplet volumes were found to agree well with the predictions of both the pendent drop weight and drop ...
Seidel SRT, Vendruscolo CP, Moreira JJ, Fülber J, Ottaiano TF, Oliva MLV, Michelacci YM, Baccarin RYA.Blood-derived autologous products are frequently used in both human and equine medicine to treat musculoskeletal disorders. These products, especially the platelet-rich plasma (PRP), may contain high concentrations of growth factors (GFs), and thus improve healing in several tissues. Nevertheless, the procedures for preparation of PRP are currently non-standardized. Several protocols, which are based on distinct centrifugation patterns (rotation speed and time), result in PRPs with different characteristics, concerning platelet and GFs concentrations, as well as platelet activation. The aim of...
Moraes CR, Runcan EE, Blawut B, Coutinho da Silva MA.The iSperm is a newly released semen analysis tool from Aidmics Biotechnology Co. LTD, which allows an iPad Mini to be transformed into a handheld microscope with objective semen analysis software for equine available through the Apple Store (version 4.5.2). The aim of this study was to compare iSperm values for sperm motility and sperm concentration to current acceptable methods for semen analysis and to determine the agreement with these methods using statistical methods. Two ejaculates from each of five Standardbred stallions were used to compare sperm motility (computer-assisted semen anal...
McClain AK, McCarrel TM.Platelet-rich plasma (PRP) is a therapeutic biologic that is used for treatment of musculoskeletal pathologies in equine athletes. Due to the expense of PRP kits, and the volumes obtained, freezing aliquots for future dosing is common. Aliquots of PRP are also commonly frozen for later analysis of growth factor concentrations in in vitro research. A variety of freezing methods are used and storage duration until analysis is often not reported. The optimal frozen storage conditions and duration to maintain concentrations of commonly measured growth factors and enzymes in PRP are unknown. Our ob...
Matczuk AK, Chodaczek G, Ugorski M.Equine arteritis virus (EAV) is a prototype member of the Arterivirus family, comprising important pathogens of domestic animals. Minor glycoproteins of Arteriviruses are responsible for virus entry and cellular tropism. The experimental methods for studying minor Arterivirus proteins are limited because of the lack of antibodies and nested open reading frames (ORFs). In this study, we generated recombinant EAV with separated ORFs 3 and 4, and Gp3 carrying HA-tag (Gp3-HA). The recombinant viruses were stable on passaging and replicated in titers similar to the wild-type EAV. Gp3-HA was incorpo...
Jiménez-Aristazábal RF, Carmona JU, Prades M.The aims of the present study were (1) to describe the microscopic and ultrastructural appearance of equine platelet-rich fibrin (PRF) clots and (2) to determine the release and degradation of transforming growth factor beta 1 (TGF-β) and insulin-like growth factor type I (IGF-I) from PRF clots incubated over 14 days. Whole blood from six horses was collected into plain tubes and centrifuged at 240 g for 8 minutes. Clots were evaluated by histology and by both transmission and scanning electronic microscopy (TEM and SEM). Growth factor concentrations were measured by ELISA at 48-hour inter...
Sander J, Terhardt M, Sander S, Aboling S, Janzen N.Up to now quantification of hypoglycin A in serum and urine in the range of nmols to μmols per liter plus the measurement of accumulated acyl conjugates have been used for the diagnosis of poisoning by fruits or seeds of in humans and animals. A second poison, methylenecyclopropylglycine, however, is known to occur in this material. The objective of our study was to develop and evaluate a method for the quantification of this compound suitable for test materials obtained from animals and man. Methods: Methylenecyclopropylglycine was extracted from serum and urine of a volunteer by a methanoli...
Serafini R, Ghosh S, Love CC, Medrano JMR, Teague SR, LaCaze KA, Varner DD.Commercially available vaginal lubricants, typically labeled as non-spermicidal, are used to lubricate equine artificial vaginas prior to semen collection. Improper type or amount of lubricant might affect stallion sperm quality, either after short-time exposure or following cooled storage of extended semen previously exposed to lubricant. The aim of this study was to evaluate stallion sperm quality following exposure to lubricant-containing extender for 1 h (T1h) or 24 h (T24h). Three ejaculates were collected from each of four stallions using a small volume of petrolatum to lubricate a...
Kamel M, Pavulraj S, Osterrieder K, Azab W.Primary infection and pathogenesis of equine herpesvirus type 1 (EHV-1) require an intricate interaction of virus with the mucosal epithelium, mononuclear cells and the vascular endothelium. Studies on EHV-1 have been facilitated by the development of different models that recapitulate the tissue complexity. The available assays can be categorized into (i) models mimicking the epithelium-peripheral blood mononuclear cell (PBMC) interaction, which include mucosal (nasal and vaginal) explants and equine respiratory epithelial cells (EREC) cultures; and (ii) PBMC-endothelium mimicking models,...
Isgren CM, Edwards T, Pinchbeck GL, Winward E, Adams ER, Norton P, Timofte D, Maddox TW, Clegg PD, Williams NJ.This study investigated changes over time in the epidemiology of extended-spectrum β-lactamase (ESBL) producing Escherichia coli within a single equine referral hospital in the UK. Faecal samples were collected from hospitalised horses in 2008 and 2017, processed using selective media and standard susceptibility laboratory methods. A novel real-time PCR with high resolution melt analysis was used to distinguish bla and bla within CTX-M-1 group. Results: In 2008, 457 faecal samples from 103 horses were collected, with ESBL-producing E. coli identified in 131 samples (28.7, 95% CI 24.6-33.1). I...
Bayless RL, Moore AR, Hassel DM, Byer BJ, Landolt GA, Nout-Lomas YS.Detection of equine acute kidney injury (AKI) is hindered by limited markers of early renal damage in horses. N-acetyl-β-D-glucosaminidase (NAG), a lysosomal enzyme in renal tubular epithelium released into urine during tubular insult, has shown promise for early identification of AKI in humans and other species. We validated an assay for NAG in equine urine and measured urinary NAG in 7 azotemic and 7 non-azotemic client-owned adult horses. The enzymatic NAG assay was validated using within- and between-run coefficients of variation (CVs), recovery following standard addition, and linearity ...
Guan F, You Y, Li X, Robinson MA.Bioactive peptides possess pharmacological effects and can be illicitly used in sports. To deter such misuse, an untargeted method using high resolution mass spectrometry (HRMS) has been developed for comprehensive detection of multitudinous exogenous peptides in equine plasma and urine. Forty-four peptides were extracted using mixed-mode solid-phase extraction (SPE) from plasma and urine, separated with a hydrophilic interaction liquid chromatography (HILIC) column, and detected on an HRMS instrument. Ammonium formate as a mobile phase additive had effects on HILIC retention and charge state ...
Boos GS, Nobach D, Failing K, Eickmann M, Herden C.A suitable RNA extraction protocol was established to gain high quality RNA from formalin-fixed paraffin-embedded tissues to perform reliable molecular assays either applicable for using FFPE tissue archives or tissues with harsh formalin-fixation. Eighteen FFPE samples from the central nervous system of horses, stored up to 11 years, were used as archive cases. To test the influence of the fixation period, brain, liver, kidney, and skeletal muscle tissue fragments from another horse, were treated either with water or tris-acetate-EDTA buffer after fixation under different timepoints with 10...
Walter J, Huwiler F, Fortes C, Grossmann J, Roschitzki B, Hu J, Naegeli H, Laczko E, Bleul U.Maturation of oocytes under in vitro conditions (IVM) results in impaired developmental competence compared to oocytes matured in vivo. As oocytes are closely coupled to their cumulus complex, elucidating aberrations in cumulus metabolism in vitro is important to bridge the gap towards more physiological maturation conditions. The aim of this study was to analyze the equine "cumulome" in a novel combination of proteomic (nano-HPLC MS/MS) and metabolomic (UPLC-nanoESI-MS) profiling of single cumulus complexes of metaphase II oocytes matured either in vivo (n = 8) or in vitro (n = 7). Re...
Hekmatynia F, Eskandarzadeh N, Imani M, Rezaei M, Zamani-Ahmadmahmudi M.Urinalysis is a critical diagnostic test which is performed in routine veterinary medicine practice. In this diagnostic test, semiquantitative measurement of urine biochemical substances is carried out using urinary dipstick. In the current study, we evaluated the diagnostic performance of human urinary dipsticks to estimate pH, specific gravity (SpG), and protein in 80 urine specimens collected from horses. These parameters were measured using two commercial human dipsticks (KP and MN in abbreviation) and quantitative reference methods. The reference methods for pH, SpG, and protein were pH m...
Nápravníková J, Petrtýl M, Stupka R, Vadlejch J.The detection and quantification of nematode eggs using fecal egg count techniques have an irreplaceable role in equine parasitic control. The reliability, particularly precision and accuracy, of individual techniques have been described only for strongylid infections. The aim of this study was to compare three fecal egg count techniques used for the detection of the two most common equine nematode infections: strongylid and ascarid. The Simple McMaster, Concentration McMaster and Mini-FLOTAC techniques were tested on spiked fecal samples with various levels of egg concentration (50, 100, 200,...
Knych HK, Baden RW, Gretler SR, McKemie DS.Despite their widespread popularity as sport and companion animals and published and anecdotal reports of vast difference in drug disposition and pharmacokinetics between individuals, studies describing equine drug metabolism are limited. It has been theorized that similar to humans, members of the CYP2D family in horses may be polymorphic in nature leading to differences in metabolism of substrates. This study aims to build on the limited current knowledge regarding P450 mediated metabolism in horses by describing the metabolism of the polymorphic CYP2D6 probe drug codeine in vitro. Codeine, ...
Sales FA, Ferreira-Silva JC, Vieira JI, Chaves MS, Caldas EL, Filho JP, Freitas VJ, Oliveira MA.Addition of extenders to thawed semen could improve fertility. Objective: To determine the efficiency of extenders to increase viability of thawed semen, measured by sperm parameters in vitro and pregnancy rates after artificial insemination (AI). Methods: Sperm motility and acrosin activity were measured during a thermoresistance test (TRT). Results: Progressive motility decreased (P<0.05) after 30 min in thawing semen treated with saline solution (SS) and only after 60 min with Tyrode's solution (TS) or freezing diluent (FD). The total motility decreased (P<0.05) after 60 min in thawed semen...
Della Malva A, De Palo P, Lorenzo JM, Maggiolino A, Albenzio M, Marino R.The aim of this study was to evaluate the effect of post-mortem aging on tenderness development and proteolysis of myofibrillar proteins in 3 different horse muscles. Warner-Bratzler shear force decreased during aging in all muscles, showing the lowest values in longissimus lumborum (LL) in all sampling points. Myofibril fragmentation index significantly increased in LL and semimembranosus (SM) muscles throughout aging time whereas in semitendinosus (ST) it increased after 14 days of aging. Proteomics analysis revealed the major content of intact myofibrillar proteins with high molecular wei...
Kandemir H, Dukik K, Hagen F, Ilkit M, Gräser Y, de Hoog GS.The anthropophilic dermatophyte Trichophyton tonsurans and its zoophilic counterpart T. equinum are phylogenetically closely related. The barcoding marker rDNA internal transcribed spacer (ITS) shows limited variation between these two species. In the current study, we combined molecular approaches with phenotypic data to determine the species boundaries between T. tonsurans (n = 52) and T. equinum (n = 15) strains originating from humans (n = 40), horses (n = 26), and a mouse (n = 1). Culture characteristics and physiology on Trichophyton agar media 1 and 5 were evaluated....
Dini P, Troch L, Lemahieu I, Deblende P, Daels P.The objective of this study was to evaluate the accuracy of a novel, portable device (iSperm Equine for assessing concentration and motility of stallion semen). In the first experiment, semen concentration was determined by the iSperm Equine (Aidmics Biotechnology), Androvision (Minitube) and NucleoCounter SP-100™ (ChemoMetec). The total motility and progressive motility were determined by the iSperm Equine and the Androvision using the manufacturer's guidelines. Frozen/thawed semen samples (n = 33) at various dilutions were analysed for concentration and motility with the above-mentioned ...
Donnelly BP, Nixon AJ, Haupt JL, Dahlgren LA.To characterize the nucleotide sequence of equine platelet-derived growth factor (PDGF)-A and -B and analyze temporal expression of these genes in equine tendon after induced tendinitis injury. Animals-18 mature horses. Methods: Genes for equine PDGF-A and -B were reverse transcribed and sequenced from synovial tissue mRNA obtained from a 3-year-old horse. Collagenase-induced lesions were created in the tensile region of the superficial digital flexor tendon in 14 horses; 3 horses served as uninjured control animals. Tendons were harvested and total RNA was isolated from experimental horses 1,...
Go YY, Wong SJ, Branscum AJ, Demarest VL, Shuck KM, Vickers ML, Zhang J, McCollum WH, Timoney PJ, Balasuriya UB.The development and validation of a microsphere immunoassay (MIA) to detect equine antibodies to the major structural proteins of equine arteritis virus (EAV) are described. The assay development process was based on the cloning and expression of genes for full-length individual major structural proteins (GP5 amino acids 1 to 255 [GP5(1-255)], M(1-162), and N(1-110)), as well as partial sequences of these structural proteins (GP5(1-116), GP5(75-112), GP5(55-98), M(88-162), and N(1-69)) that constituted putative antigenic regions. Purified recombinant viral proteins expressed in Escherichia col...
Kjaer LN, Lungholt MM, Nielsen MK, Olsen SN, Maddox-Hyttel C.Increased knowledge is needed to assist in the interpretation of presently available diagnostic techniques for infection by the tapeworm Anoplocephala perfoliata in horses. Objective: The suggested cut-off level of an A. perfoliata specific ELISA may not adequately reflect the actual infection level. Hence, faecal egg counts may be a more useful diagnostic test for individual horses than previously reported. Methods: Eighty-four horses admitted for slaughter at a Danish abattoir were examined for the presence of A. perfoliata. The number of tapeworms, their stage of development and gross patho...
Cain JL, Slusarewicz P, Rutledge MH, McVey MR, Wielgus KM, Zynda HM, Wehling LM, Scare JA, Steuer AE, Nielsen MK.Fecal egg counts are the cornerstone of equine parasite control programs. Previous work led to the development of an automated, image-analysis-based parasite egg counting system. The system has been further developed to include an automated reagent dispenser unit and a custom camera (CC) unit that generates higher resolution images, as well as a particle shape analysis (PSA) algorithm and machine learning (ML) algorithm. The first aim of this study was to conduct a comprehensive comparison of method precision between the original smartphone (SP) unit with the PSA algorithm, CC/PSA, CC/ML, and ...
Mugnier S, Kervella M, Douet C, Canepa S, Pascal G, Deleuze S, Duchamp G, Monget P, Goudet G.Oviduct epithelial cells (OEC) co-culture promotes in vitro fertilization (IVF) in human, bovine and porcine species, but no data are available from equine species. Yet, despite numerous attempts, equine IVF rates remain low. Our first aim was to verify a beneficial effect of the OEC on equine IVF. In mammals, oviductal proteins have been shown to interact with gametes and play a role in fertilization. Thus, our second aim was to identify the proteins involved in fertilization in the horse. Results: In the first experiment, we co-incubated fresh equine spermatozoa treated with calcium ionophor...
Grubaugh ND, Petz LN, Melanson VR, McMenamy SS, Turell MJ, Long LS, Pisarcik SE, Kengluecha A, Jaichapor B, O'Guinn ML, Lee JS.Highly multiplexed assays, such as microarrays, can benefit arbovirus surveillance by allowing researchers to screen for hundreds of targets at once. We evaluated amplification strategies and the practicality of a portable DNA microarray platform to analyze virus-infected mosquitoes. The prototype microarray design used here targeted the non-structural protein 5, ribosomal RNA, and cytochrome b genes for the detection of flaviviruses, mosquitoes, and bloodmeals, respectively. We identified 13 of 14 flaviviruses from virus inoculated mosquitoes and cultured cells. Additionally, we differentiate...
Yasui M, Suzuki N, Liu X, Okamoto Y, Kim SY, Laxmi YR, Shibutani S.4-Hydroxyequilenin (4-OHEN)-dC is a major, potentially mutagenic DNA adduct induced by equine estrogens used for hormone replacement therapy. To study the miscoding property of 4-OHEN-dC and the involvement of Y-family human DNA polymerases (pols) eta, kappa and iota in that process, we incorporated 4-OHEN-dC into oligodeoxynucleotides and used them as templates in primer extension reactions catalyzed by pol eta, kappa and iota. Pol eta inserted dAMP opposite 4-OHEN-dC, accompanied by lesser amounts of dCMP and dTMP incorporation and base deletion. Pol kappa promoted base deletions as well as ...
Smith RK, Heinegård D.Cartilage oligomeric matrix protein (COMP) is a noncollagenous extracellular matrix protein found predominantly in cartilage, but also in tendon, ligament and meniscus. Studies in man have demonstrated that it may be used as a prognostic marker in rheumatoid arthritis and osteoarthritis. The present study investigated whether tendon injury contributes to serum and tendon sheath synovial fluid levels of COMP in horses. COMP levels, analysed by competitive ELISA, in the digital sheath synovial fluid were more than 10-fold higher than in the serum. Levels were significantly raised when tendon dam...
Raphel CF, Beech J.Of 122 horses with pleural effusion, 90 (73.8%) had pleuritis secondary to pneumonia or lung abscessation. Fifty-one horses died or were euthanatized. The highest prevalence was in Thoroughbred and Standardbred racehorses. Eleven (12.2%) horses were postsurgical patients and 22 (24.4%) horses had been transported over 500 miles. There was no relationship between final outcome and the age, sex, breed, hematologic values, or laboratory findings pertaining to pleural fluid except for the bacterial isolation of Escherichia coli from the pleural fluid, as this was more frequently associated with de...
Noronha LE, Harman RM, Wagner B, Antczak DF.The low-affinity Fc receptor CD16 plays a central role in the inflammatory and innate immune responses of many species, but has not yet been investigated in the horse. Using the predicted extracellular region of equine CD16 expressed as a recombinant fusion protein with equine IL-4 (rIL-4/CD16), we generated a panel of mouse monoclonal antibodies (mAbs) that recognize equine CD16. Nine mAbs were chosen for characterization based upon recognition of CD16, but not IL-4, in ELISA. All nine mAbs recognized full-length, cell-surface CD16 expressed as a GFP fusion protein by CHO cells, but not the c...
LaFrentz S, Abarca E, Mohammed HH, Cuming R, Arias CR.The equine conjunctival microbiota has often been reported to be dominated by Gram-positive species such as Staphylococcus sp., Bacillus sp., and Corynebacterium sp. However, traditional culture-based methods can only recover a fraction of the bacterial species present in the sample. Objective: This pilot study aimed at exploring the diversity of the equine conjunctival microbiota using culture-independent methods. Methods: Eight horses were included in this study, and only eyes with normal ophthalmic examination (n = 15 eyes) were sampled. Methods: Conjunctival biopsies (culture-independent...
Fessler JF, Bottoms GD, Coppoc GL, Gimarc S, Latshaw HS, Noble JK.Endotoxin (LPS) was quantitated in experimental subjects and in horses with naturally occurring gastrointestinal strangulation obstruction and/or septicaemic diseases to establish the fate of LPS and the clinical usefulness of the Limulus amoebocyte lysate (LAL) assay. The assay was validated for sensitivity (10 pg/ml), recovery (90 to 106 per cent), intra-assay precision (CV = 5.5 per cent) inter-assay precision (CV = 11 per cent), and stability of diluted, heat treated, frozen samples (at least 90 days). Plasma concentrations of LPS after sublethal (3 micrograms/kg) jugular or portal vein bo...
Cheadle MA, Lindsay DS, Rowe S, Dykstra CC, Williams MA, Spencer JA, Toivio-Kinnucan MA, Lenz SD, Newton JC, Rolsma MD, Blagburn BL.An IFAT was used to determine the prevalence of Neospora-specific IgG antibodies in serum from Alabama horses. Serum samples (n = 536) were from asymptomatic horses routinely submitted for equine infectious anaemia virus infection testing. We also subjected a 13-year-old horse with CNS disease to necropsy examination for isolation and in vitro cultivation of protozoal organisms. In antemortem tests, this horse was positive for antibodies to Neospora sp. in the IFAT and western immunoblot. Results of the prevalence survey indicated that IgG antibodies to Neospora were present in 62 (11.5%) of t...
Morozova-Roche LA, Arico-Muendel CC, Haynie DT, Emelyanenko VI, Van Dael H, Dobson CM.Native state 1H NMR resonance assignments for 125 of the 129 residues of equine lysozyme have enabled measurement of the hydrogen exchange kinetics for over 60 backbone amide and three tryptophan indole hydrogen atoms in the native state. Native holo equine lysozyme hydrogen exchange protection factors are as large as 10(6), the most protected residues being located in elements of secondary structure. High exchange protection in the domain interface correlates with the binding of Ca2+ in this region. Equine lysozyme differs from most non-Ca2+ binding lysozymes in forming a highly populated par...
Reubel GH, Barlough JE, Madigan JE.We report on the production and characterization of Ehrlichia risticii, the agent of Potomac horse fever (PHF), from snails (Pleuroceridae: Juga spp.) maintained in aquarium culture and compare it genetically to equine strains. Snails were collected from stream waters on a pasture in Siskiyou County, Calif., where PHF is enzootic and were maintained for several weeks in freshwater aquaria in the laboratory. Upon exposure to temperatures above 22 degrees C the snails released trematode cercariae tentatively identified as virgulate cercariae. Fragments of three different genes (genes for 16S rRN...
Ball BA, Almeida J, Conley AJ.Endocrinological assays are important for evaluation of mares with granulosa-cell tumours (GCTs), and our research in mares indicates that anti-Müllerian hormone (AMH) may be a good biomarker for this type of ovarian tumour. Objective: To evaluate the use of serum AMH concentrations for endocrine diagnosis of GCTs in mares. Methods: Archived serum samples (n = 403) previously assayed for determination of serum inhibin, testosterone and progesterone concentrations (GCT panel) were assayed for serum AMH concentrations using a heterologous enzyme-linked immunosorbent assay previously validated b...
Dini P, El-Sheikh Ali H, Carossino M, C Loux S, Esteller-Vico A, E Scoggin K, Daels P, A Ball B.Equine chromosome 24 microRNA cluster (C24MC), the ortholog of human C14MC, is a pregnancy-related miRNA cluster. This cluster is believed to be implicated in embryonic, fetal, and placental development. The current study aimed to characterize the expression profile of this cluster in maternal circulation throughout equine gestation. The expression profile of miRNAs belonging to this cluster was analyzed in the serum of non-pregnant (diestrus), pregnant (25 d, 45 d, 4 mo, 6 mo, 10 mo), and postpartum mares. Among the miRNAs examined, 11 miRNAs were differentially expressed across the analyzed ...
Kingston JK, Bayly WM, Sellon DC, Meyers KM, Wardrop KJ.To investigate the effects of sodium citrate, low molecular weight heparin (LMWH), and prostaglandin E1 (PGE1) on aggregation, fibrinogen binding, and enumeration of equine platelets. Methods: Blood samples obtained from 4 Thoroughbreds. Methods: Blood was collected into syringes in the ratio of 9 parts blood:1 part anticoagulant. Anticoagulants used were sodium citrate, LMWH, sodium citrate and LMWH, or 300 nM PGE1/ml of anticoagulant. Platelet aggregation in response to ADP, collagen, and PGE1 was assessed, using optical aggregometry. Platelet activation was evaluated, using flow cytometry, ...
Johnson AL, McAdams-Gallagher SC, Aceto H.The laboratory diagnosis of botulism in horses traditionally has relied upon the mouse bioassay (MBA). The accuracy of this test for the diagnosis of botulism in horses is unknown. Objective: Our goal was to determine the sensitivity, specificity, positive predictive value, and negative predictive value of the MBA on laboratory-processed fecal and gastrointestinal samples for foals and adult horses. Methods: Cases included all horses with a final clinical diagnosis of botulism that were admitted between 1986 and 2011 and had MBA testing performed. Controls included horses without botulism that...
Bundgaard L, Åhrman E, Malmström J, Auf dem Keller U, Walters M, Jacobsen S.The objectives of this study was to establish a sensitive and reproducible method to map the cartilage and subchondral bone proteomes in quantitative terms, and mine the proteomes for proteins of particular interest in the pathogenesis of osteoarthritis (OA). The horse was used as a model animal. Protein was extracted from articular cartilage and subchondral bone samples from three horses in triplicate by pressure cycling technology or ultrasonication. Digested proteins were analysed by data independent acquisition based mass spectrometry. Data was processed using a pre-established spectral li...
Bleumink-Pluym NM, Werdler ME, Houwers DJ, Parlevliet JM, Colenbrander B, van der Zeijst BA.A PCR for the detection of Taylorella equigenitalis, the causative agent of contagious equine metritis, was developed and evaluated. A genus-specific primer-probe set was derived from the 16S ribosomal DNA sequences. The PCR was specific and amplified a 585-bp product from all 64 available T. equigenitalis isolates. This PCR product hybridized with a specific probe in a dot spot assay. A variety of microorganisms from the genital tracts of horses or with a close phylogenetic relationship to T. equigenitalis did not yield a visible PCR product and were all negative in the dot spot hybridization...
Brown K, Moreton J, Malla S, Aboobaker AA, Emes RD, Tarlinton RE.The recently released draft horse genome is incompletely characterised in terms of its repetitive element profile. This paper presents characterisation of the endogenous retrovirus (ERVs) of the horse genome based on a data-mining strategy using murine leukaemia virus proteins as queries. 978 ERV gene sequences were identified. Sequences were identified from the gamma, epsilon and betaretrovirus genera. At least one full length gammaretroviral locus was identified, though the gammaretroviral sequences are very degenerate. Using these data the RNA expression of these ERVs were derived from RNA ...
Darlington RW, James C.Darlington, R. W. (St. Jude Children's Research Hospital, Memphis, Tenn.), and C. James. Biological and morphological aspects of the growth of equine abortion virus. J. Bacteriol. 92:250-257. 1966.-The growth of equine abortion virus (EAV) was studied by bioassay and electron microscopy in L-cell monolayer and suspension cultures, and in HeLa and BHK 21/13 cell monolayers. Results of virus assay (plaque-forming units) indicated that production of cell-associated virus (CAV) began at 6 to 9 hr after infection in all of the cell strains used. Virus release occurred 1 to 2 hr later. By 15 to 20 h...
Hildreth SW, Beaty BJ, Maxfield HK, Gilfillan RF, Rosenau BJ.Enzyme immunoassays (EIAs) for eastern equine encephalomyelitis (EEE) and Highlands J (HJ) virus antigens were compared in a retrospective study with standard virus isolation procedures (VIP) for detection of alpha virus-infected mosquito pools. The original VIP was a plaque assay in chick embryo cell culture, and was performed in the years from 1979 to 1981. Using the original VIP as the reference standard, the sensitivity rate of the EIA was 0.7674 and the false negative rate was 0.2326. However, when the storage age and the initial virus titer of the sample were considered, the sensitivity ...
Zevenhoven-Dobbe JC, Greve S, van Tol H, Spaan WJM, Snijder EJ.Equine arteritis virus (EAV) contains seven structural proteins that are all required to produce infectious progeny. Alphavirus-based expression vectors have been generated for each of these proteins to explore the possibilities for their constitutive expression in cell lines. This approach was successful for minor glycoproteins GP(2b), GP(3) and GP(4) and for the E protein. Subsequently, it was demonstrated that cell lines expressing these proteins could rescue EAV mutants that were disabled in the expression of the corresponding gene, resulting in the production of virus particles carrying t...
Trumble TN, Trotter GW, Oxford JR, McIlwraith CW, Cammarata S, Goodnight JL, Billinghurst RC, Frisbie DD.To determine concentrations of matrix metalloproteinase (MMP)-2 and -9 in synovial fluid; and mRNA expression of MMP-1, -13, and -3; interleukin[IL]-1alpha and beta; and tumor necrosis factor (TNF)-alpha in synovial membrane and articular cartilage from horses with naturally occurring joint disease. Methods: Synovial fluid (n = 76), synovial membrane (59), and articular cartilage (45) from 5 clinically normal horses and 55 horses with joint disease categorized as traumatic (acute [AT] or chronic [CT]), osteochondritis dissecans (OCD), or septic (S). Methods: Synovial fluid gelatinase concentra...
Kim SK, Kim S, Dai G, Zhang Y, Ahn BC, O'Callaghan DJ.The equine herpesvirus 1 (EHV-1) negative regulatory IR2 protein (IR2P), an early 1,165-amino acid (aa) truncated form of the 1487-aa immediate-early protein (IEP), lacks the trans-activation domain essential for IEP activation functions but retains domains for binding DNA, TFIIB, and TBP and the nuclear localization signal. IR2P mutants of the N-terminal region which lack either DNA-binding activity or TFIIB-binding activity were unable to down-regulate EHV-1 promoters. In EHV-1-infected cells expressing full-length IR2P, transcription and protein expression of viral regulatory IE, early EICP...
Lawrence GL, Gilkerson J, Love DN, Sabine M, Whalley JM.Sets of primers were designed which enabled specific amplification of homologous regions of the glycoprotein C and gene 76 genetic loci of equine herpesviruses 1 and 4 (EHV-1 and EHV-4). The resultant virus-specific polymerase chain reaction (PCR) products arising from each loci could be discriminated easily on the basis of size on an agarose gel, allowing rapid differentiation of the two equine herpesviruses. Specificity of the amplifications were confirmed by Southern hybridization and restriction endonuclease digestion. The PCR test was applied to nasal swab samples from weanling foals and ...
Guan F, Uboh C, Soma L, Hess A, Luo Y, Tsang DS.Beclomethasone dipropionate (BDP) is a potent pro-drug to beclomethasone (BOH) and is used in the treatment of chronic and acute respiratory disorders in the horse. The therapeutic dose of BDP (325 microg per horse) by inhalation results in very low plasma and urinary concentrations of BDP and its metabolites that pose a challenge to detection and confirmation by equine forensic laboratories. To solve this problem, a method involving the use of a liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) was developed for the detection, confirmation and quantification of the analyt...
Read JE, Cabrera-Sharp V, Offord V, Mirczuk SM, Allen SP, Fowkes RC, de Mestre AM.Equine chorionic girdle trophoblast cells play important endocrine and immune functions critical in supporting pregnancy. Very little is known about the genes and pathways that regulate chorionic girdle trophoblast development. Our aim was to identify genes and signalling pathways active in vivo in equine chorionic girdle trophoblast within a critical 7-days window. We exploited the late implantation of the equine conceptus to obtain trophoblast tissue. An Agilent equine 44K microarray was performed using RNA extracted from chorionic girdle and chorion (control) from equine pregnancy days 27, ...
