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.
Baj A, Beltramini GA, Romano M, Lauritano D, Gaudio RM, Palmieri A, Cura F, Giannì AB.BIOPAD® is an ivory-white soft sponge, made exclusively of lyophilized type I native heterologous collagen extracted from horse flexor tendon, gelatine free, that keeps its native structure specific to the body’s skin tissue. BIOPAD® is an active dressing, playing an active role in all stages of wound healing process, stimulating granulation tissue growth and enhancing regeneration tissues. It ensures balance between absorption and humidity at wound surface, gaseous exchange of soft tissues during healing process, barrier to prevent bacterial infections and it is completely non-adherent. T...
Tangyuenyong S, Nambo Y, Nagaoka K, Tanaka T, Watanabe G.Most thyroid hormone determinations in animals are based on immunoassays adapted from those used to test human samples, which may not reflect the actual values of thyroid hormone in horses because of the presence of binding proteins. The aims of the present study were i) to establish a novel radioimmunoassay (RIA) using a more simple and convenient method to separate binding proteins for the measurement of total thyroxine (T4) in horses and ii) to validate the assay by comparing total T4 concentrations in yearling horses raised in different climates. Blood samples were collected from trained y...
Lepczyński A, Ożgo M, Dratwa-Chałupnik A, Robak P, Pyć A, Zaborski D, Herosimczyk A.The main objectives of the study were to: (1) deeply analyse the serum protein composition of Equus caballus, (2) assess the effectiveness of the high-abundant protein depletion and improve the concentration of medium- and low-abundant proteins. The analysis were performed on the blood plasma of three healthy part-Arabian mares. The implementation of two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation - time of flight mass spectrometry allowed us to establish a horse plasma proteome map. Serum proteins were resolved at pH 4 to 7, followed by 12% SDS-PAGE. As a resul...
González-Fernández L, Sánchez-Calabuig MJ, Alves MG, Oliveira PF, Macedo S, Gutiérrez-Adán A, Rocha A, Macías-García B.Equine cumulus-oocyte complexes (COCs) are classified as compact (cCOC) or expanded (eCOC) and vary in their meiotic competence. This difference could be related to divergent glucose metabolism. To test this hypothesis in the present study, eCOCs, cCOCs and expanded or compact mural granulosa cells (EC and CC respectively) were matured in vitro for 30h, at which time maturation rate, glucose metabolism and the expression of genes involved in glucose transport, glycolysis, apoptosis and meiotic competence were determined. There were significant differences between eCOCs and cCOCs in maturation ...
Dohre SK, Kamthan A, Singh S, Alam SI, Kumar S.Glanders is a disease of horses, donkeys and mules. The causative agent Burkholderia mallei, is a biorisk group 3 pathogen and is also a biothreat agent. Simple and rapid diagnostic tool is essential for control of glanders. Using a proteomic approach and immunoblotting with equine sera, we identified 12 protein antigens that may have diagnostic potential. Various immunoreactive proteins e.g. GroEL, translation elongation factor Tu, elongation factor Ts, arginine deiminase, malate dehydrogenase, DNA directed RNA polymerase subunit alpha were identified on 2-dimentional immunoblots. One of thes...
Scott EY, Mansour T, Bellone RR, Brown CT, Mienaltowski MJ, Penedo MC, Ross PJ, Valberg SJ, Murray JD, Finno CJ.Efforts to resolve the transcribed sequences in the equine genome have focused on protein-coding RNA. The transcription of the intergenic regions, although detected via total RNA sequencing (RNA-seq), has yet to be characterized in the horse. The most recent equine transcriptome based on RNA-seq from several tissues was a prime opportunity to obtain a concurrent long non-coding RNA (lncRNA) database. This lncRNA database has a breadth of eight tissues and a depth of over 20 million reads for select tissues, providing the deepest and most expansive equine lncRNA database. Utilizing the intergen...
Harman RM, Yang S, He MK, Van de Walle GR.The prevalence of chronic skin wounds in humans is high, and treatment is often complicated by the presence of pathogenic bacteria. Therefore, safe and innovative treatments to reduce the bacterial load in cutaneous wounds are needed. Mesenchymal stromal cells (MSC) are known to provide paracrine signals that act on resident skin cells to promote wound healing, but their potential antibacterial activities are not well described. The present study was designed to examine the antibacterial properties of MSC from horses, as this animal model offers a readily translatable model for MSC therapies i...
Zak A, Siwinska N, Slowikowska M, Borowicz H, Kubiak K, Hildebrand J, Popiolek M, Niedzwiedz A.There are no available studies describing the possible resistance of strongyles to ivermectin in horses in Poland. One hundred seventy three horses from nine stud farms from South-Western Poland were studied. The effectiveness of ivermectin was studied on the 14th day after ivermectin administration using the fecal egg count reduction test, and a long-term observation of the egg reappearance period was carried out. The fecal study was carried out using a modified McMaster method, which typically detects 20 eggs per gram of stool. The results were subjected to statistical analysis that enabled ...
Dube S, Chionuma H, Matoq A, Alshiekh-Nasany R, Abbott L, Poiesz BJ, Dube DK.In order to better understand the training and athletic activity of horses, we must have complete understanding of the isoform diversity of various myofibrillar protein genes like tropomyosin. Tropomyosin (TPM), a coiled-coil dimeric protein, is a component of thin filament in striated muscles. In mammals, four TPM genes (TPM1, TPM2, TPM3, and TPM4) generate a multitude of TPM isoforms via alternate splicing and/or using different promoters. Unfortunately, our knowledge of TPM isoform diversity in the horse is very limited. Hence, we undertook a comprehensive exploratory study of various TPM i...
Jiménez-Aristizabal RF, López C, Álvarez ME, Giraldo C, Prades M, Carmona JU.To compare the temporal release (over three weeks) of tumor necrosis factor alpha (TNF-α), interleukin 4 (IL-4), IL-1 receptor antagonist (IL-1ra), platelet-derived growth factor BB (PDGF-BB) and transforming growth factor beta-1 (TGF-β1) from two platelet-rich fibrin (PRF) preparations from equine blood obtained at either 240g/8min or 416g/10min. Whole blood from 10 horses was used to obtain PRF clots by two different centrifugation protocols. After 1h of rest, PRF clots were deposited in wells with culture medium, which was changed at 6h, 24h and then every 48h to 21days. Cytokines and GFs...
Ashraf Kharaz Y, Zamboulis D, Sanders K, Comerford E, Clegg P, Peffers M.Exploring the tendon proteome is a challenging but important task for understanding the mechanisms of physiological/pathological processes during ageing and disease and for the development of new treatments. Several extraction methods have been utilised for tendon mass spectrometry, however different extraction methods have not been simultaneously compared. In the present study we compared protein extraction in tendon with two chaotropic agents, guanidine hydrochloride (GnHCl) and urea, a detergent, RapiGest™, and their combinations for shotgun mass spectrometry. An initial proteomic analysi...
Popot MA, Ho ENM, Stojiljkovic N, Bagilet F, Remy P, Maciejewski P, Loup B, Chan GHM, Hargrave S, Arthur RM, Russo C, White J, Hincks P, Pearce C....Cobalt is an essential mineral micronutrient and is regularly present in equine nutritional and feed supplements. Therefore, cobalt is naturally present at low concentrations in biological samples. The administration of cobalt chloride is considered to be blood doping and is thus prohibited. To control the misuse of cobalt, it was mandatory to establish an international threshold for cobalt in plasma and/or in urine. To achieve this goal, an international collaboration, consisting of an interlaboratory comparison between 5 laboratories for the urine study and 8 laboratories for the plasma stud...
Strauch S, Wichert B, Greef JM, Hillegeist D, Zeyner A, Liesegang A.The aim of this study was to improve an in vitro system in order to gather optimized information on the digestion of different forages in the horse's upper gastrointestinal tract. Therefore, foregut digestion of several forages was simulated in vitro (Part 1). The effect of different pH values on in vitro fructan degradation of two selected grasses (Part 2) was tested subsequently. Part 1: We hypothesized that our system produces representative results simulating digestive processes in the upper alimentary tract, but neglects microbial fermentation. In vitro digestion of six forages (grass ...
Bastos FZ, Barussi FCM, Santi TF, Vieira BP, Senegaglia AC, Cruz FF, Michelotto PV.There is no consensus on aspects of equine bone marrow collection and processing. The study aimed to describe the collection of large volumes of bone marrow from horses of advanced age, with emphasis on bone marrow mononuclear cells (BMMCs) recovery and viability after cryopreservation. Fourteen horses, aged 3-24 years, were divided into three experiments. E1 studied the feasibility of collecting 200 mL from the sternums of horses of advanced age; E2 examined the number of cells obtained from the first and last syringe of each puncture; and E3 investigated the influence of heparin concentrati...
Shin HD, Suh JH, Kim J, Cho HD, Lee SD, Han KS, Wang Y, Han SB.A high throughput method for simultaneous screening of anabolic steroids and their metabolites (4-esterendione, trenbolone, boldenone, oxandrolone, nandrolone, methandrostenolone, testosterone, 1-androstendione, ethisterone, normethandrolone, methyltestosterone, 16β-Hydroxystanozolol, epitestosterone, bolasterone, norethandrolone, danazol, stanozolol and androstadienone) in equine urine by online turbulent flow extraction coupled with liquid chromatography-tandem mass spectrometry was developed. The use of turbulent flow chromatography could simplify pretreatment of horse urine, which has com...
Torgeman A, Mador N, Dorozko M, Lifshitz A, Eschar N, White MD, Wolf DG, Epstein E.Assuring viral safety of horse plasma-derived products is fundamental for ethical and regulatory reasons. We previously demonstrated the ability of pepsin digestion at low pH to inactivate West Nile and Sindbis viruses in horse plasma. The present study further examined the efficiency of pepsin digestion to inactivate four additional viruses: HSV-1 and BVDV (lipid-enveloped), BPV and Reo-3 (nonenveloped). These viruses were spiked into hyperimmunized horse plasma against botulinum toxin and subjected to low pH (3.2) alone or combined with pepsin digestion (1200 units/ml). Peptic digestion inac...
Dimsoski P.To show that application of the polymerase chain reaction (PCR) method modified for amplification of a low-copy number DNA samples, ie, the isolation of PCR products (IPCRp), would represent improvement in obtaining genotypes from a fecal DNA compared with previously used genotyping methods. Methods: The DNA from the horse fecal matter was extracted by modified Qiagen DNA Stool Mini Kit protocol. Following the extraction, the DNA genotypes from fecal samples were obtained by the most powerful PCR amplification method, the IPCRp. The IPCRp-based multiplex kit amplified biotin-labeled strands we...
Monk CS, Jeong SY, Gibson DJ, Plummer CE.Tetracyclines have activity against matrix metalloproteinases (MMP). Oral medications with effects on the ocular surface are of interest in patients where repeated topical dosing is limited. The aim of this study was to characterize the concentration of minocycline in the tears of normal horses after oral administration and to determine if this level directly inhibits MMP activity. Methods: Five healthy adult ponies were administered oral minocycline (Wedgewood Pharmacy; Swedesboro, NJ) at 4 mg/kg every 12 h for 5 days. Tears were collected at T = 2, 26, 50, 56, 74, 80, and 98 h. Tear minocycl...
Wong JKY, Leung DKK, Curl P, Schiff PJ, Lam KKH, Wan TSM.The detection of boldenone, nandrolone, 5(10)-estrene-3β,17α-diol, and 4-estrene-3,17-dione in a urine sample collected from a gelding having been treated with testosterone (500 mg 'Testosterone Suspension 100', single dose, injected intramuscularly) in 2009 led the authors' laboratory to suspect that these 'testicular' steroids could be minor metabolites of testosterone in geldings. Administration trials on six castrated horses with Testosterone Suspension 100 confirmed that low levels of boldenone, nandrolone, 5(10)-estrene-3β,17α-diol, and 4-estrene-3,17-dione could indeed be detected ...
Ujvari S, Schwarzwald CC, Fouché N, Howard J, Schoster A.Point-of-care (POC) diagnostic tests with good sensitivity and specificity are needed for diagnosing failure of transfer of passive immunity (FTPI) in foals. Turbidimetric immunoassays (TIA) have these characteristics and provide quantitative results. A commercially available TIA-based POC test (POC-TIA) has not been validated in horses. Objective: To validate a POC-TIA and compare results of POC-TIA, a POC-ELISA, and radial immunodiffusion (RID). Methods: Heparinized blood samples (n = 127) from 48 hospitalized foals (<12 hour to 48 days). Methods: Prospective validation study. IgG concentrat...
Bailly-Chouriberry L, Baudoin F, Cormant F, Glavieux Y, Loup B, Garcia P, Popot MA, Bonnaire Y.The improvement of doping control is an ongoing race. Techniques to fight doping are usually based on the direct detection of drugs or their metabolites by analytical methods such as chromatography hyphenated to mass spectrometry after ad hoc sample preparation. Nowadays, omic methods constitute an attractive development and advances have been achieved particularly by application of molecular biology tools for detection of anabolic androgenic steroids (AAS), erythropoiesis-stimulating agent (ESA), or to control human growth hormone misuses. These interesting results across different animal spe...
Wong JKY, Kwok WH, Chan GHM, Choi TLS, Ho ENM, Jaubert M, Bailly-Chouriberry L, Bonnaire Y, Cawley A, Ming Williams H, Keledjian J, Brooks L....Acadesine, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, commonly known as AICAR, is a naturally occurring adenosine monophosphate-activated protein kinase (AMPK) activator in many mammals, including humans and horses. AICAR has attracted considerable attention recently in the field of doping control because of a study showing the enhancement of endurance performance in unexercised or untrained mice, resulting in the term 'exercise pill'. Its use has been classified as gene doping by the World Anti-Doping Agency (WADA), and since it is endogenous, it may only be possible to control del...
Pihl TH, Jacobsen S, Olsen DT, Højrup P, Grosche A, Freeman DE, Andersen PH, Houen G.OBJECTIVE To purify and characterize equine vitamin D-binding protein (VDBP) from equine serum and to evaluate plasma concentrations of VDBP in healthy horses and horses with gastrointestinal injury or disease. ANIMALS 13 healthy laboratory animals (8 mice and 5 rabbits), 61 healthy horses, 12 horses with experimentally induced intestinal ischemia and reperfusion (IR), and 59 horses with acute gastrointestinal diseases. PROCEDURES VDBP was purified from serum of 2 healthy horses, and recombinant equine VDBP was obtained through a commercial service. Equine VDBP was characterized by mass spectr...
Frank N, Walsh DM.Repeatability of the oral sugar test (OST) has not been evaluated. Objective: We hypothesized that OST glucose, insulin, active (aGLP-1) and total (tGLP-1) glucagon-like peptide 1, and high-molecular-weight (HMW) adiponectin results would be repeatable. Methods: Fifty-three horses from a Tennessee research facility (n = 23) and private practice in Missouri (n = 30), including animals with medical histories of equine metabolic syndrome. Methods: Two OSTs were performed 7-14 days apart and plasma glucose and insulin concentrations were measured at 0, 60, and 75 minutes; a positive result w...
Collas C, Sallé G, Dumont B, Cabaret J, Cortet J, Martin-Rosset W, Wimel L, Fleurance G.The spread of anthelmintic resistance in equine strongyle nematodes has become a major problem, advocating for the development of alternative control for strongyles. Our study consisted of both in vivo and in vitro experiments. We investigate for the first time the efficacy of a short-term consumption of tannin-rich sainfoin (Onobrychis viciifolia) or extra proteins in naturally infected horses. We used 30 horses allocated into three groups of 10 individuals that received for 18 days either (i) a tannin-rich diet with 70% DM sainfoin pellets (Sd), (ii) a protein-rich diet with 52% DM Italian r...
Shah YY, Maldonado-Camargo L, Patel NS, Biedrzycki AH, Yarmola EG, Dobson J, Rinaldi C, Allen KD.The mechanics of synovial fluid vary with disease progression, but are difficult to quantify quickly in a clinical setting due to small sample volumes. In this study, a novel technique to measure synovial fluid mechanics using magnetic nanoparticles is introduced. Briefly, microspheres embedded with superparamagnetic iron oxide nanoparticles, termed magnetic particles, are distributed through a 100μL synovial fluid sample. Then, a permanent magnet inside a protective sheath is inserted into the synovial fluid sample. Magnetic particles translate toward the permanent magnet and the percentage ...
Perez de Souza L, Naake T, Tohge T, Fernie AR.The grand challenge currently facing metabolomics is the expansion of the coverage of the metabolome from a minor percentage of the metabolic complement of the cell toward the level of coverage afforded by other post-genomic technologies such as transcriptomics and proteomics. In plants, this problem is exacerbated by the sheer diversity of chemicals that constitute the metabolome, with the number of metabolites in the plant kingdom generally considered to be in excess of 200 000. In this review, we focus on web resources that can be exploited in order to improve analyte and ultimately metabol...
Guan M, Dai D, Li L, Wei J, Yang H, Li S, Zhang Y, Lin Y, Xiong S, Zhao Z.High levels of triacylglycerols (TGs) have been linked to cardiovascular disease and liver diseases. Comprehensively analyzing TGs is helpful to understand the TGs functions in these diseases. However, due to the existence of a large number of isomers TGs and the lack of commercial standards, precise analysis of individual triacylglycerol (TG) with specific fatty acid chain composition is full of challenge. In this work, ultra-performance liquid chromatography (UPLC) coupled with electrospray ionization (ESI) mass spectrometry (MS) were employed for comprehensive qualification and quantificati...
Thane K, Bedenice D, Pacheco A.To determine the extent of intraoperator (between duplicate samples) and interoperator (between different operators) variability in equine thromboelastography (TEG). Methods: Kaolin-activated TEG was performed in duplicate by operator-pair A/B or A/C (2 groups of 10 horses) using discrete setups, within 30-45 minutes of vacuum-assisted blood collection. Recorded TEG variables included clot initiation time (R), clot formation time (K), rate of clot formation (α), clot strength (MA), and viscoelastic/shear strength (G). Operators independently determined the clinical coagulation status for each...
Boelow H, Krücken J, Thomas E, Mirams G, von Samson-Himmelstjerna G.Due to high prevalence of anthelmintic resistance in equine helminths, selective treatment is increasingly promoted and in some countries a positive infection diagnosis is mandatory before treatment. Selective treatment is typically recommended when the number of worm eggs per gram faeces (epg) exceeds a particular threshold. In the present study we compared the semi-quantitative sedimentation/flotation method with the quantitative methods Mini-FLOTAC and FECPAK in terms of precision, sensitivity, inter-rater reliability and correlation of worm egg counts to improve the choice of optimal diagn...
Masri M, Lombardero G, Velasquillo C, Martínez V, Neri R, Villegas H, Ibarra C.The goal of this study is to evaluate the efficiency of obtaining a large number of viable cells within a construct that will not be detached by high fluid flow during arthroscopic implantation. Methods: Arthroscopic osteochondral biopsy specimens were obtained from the medial femoral trochlea of 8 horses. Chondrocytes were isolated by collagenase digestion and expanded in M199 media until confluency. After 10 to 12 days, cultures were trypsinized and cells resuspended in culture media. Then, 5 x 10(6) cells x mL(-1) were seeded on a culture dish and the same amount in a flask. Once extracellu...
Knych HK, Vidal MA, Casbeer HC, McKemie DS.The use of triamcinolone acetonide (TA) in performance horses necessitates establishing appropriate withdrawal times prior to performance. Objective: To describe the plasma pharmacokinetics of TA and time-related urine and synovial fluid concentrations following i.m. and intra-articular administration to exercised Thoroughbred horses. Methods: Block design. Methods: Twelve racing fit adult Thoroughbred horses received a single i.m. administration of TA (0.1 mg/kg bwt). After an appropriate washout period, the same horses then received a single intra-articular TA administration (9 mg) into the ...
Li LF, Guan WJ, Hua Y, Bai XJ, Ma YH.A fibroblast line was successfully established from Mongolian horse ear marginal tissue by using a primary explant technique and cell cryogenic preservation technology. Biological analysis showed the following: The cells were adherent and exhibited density-dependent inhibition of proliferation; assays of microbial contamination from bacteria, fungi, and mycoplasma were negative; the population doubling time of the cells was 33.9 h; and a 2n chromosome number of 64 at a frequency higher than 80%. A lack of cross-contamination of this cell line with other species was confirmed by isoenzyme analy...
Brandon CI, Heusner GL, Caudle AB, Fayrer-Hosken RA.The objectives of this study were to 1) identify proteins found in stallion seminal plasma utilizing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in conjunction with Western blot analysis; and 2) to determine if any of these individual proteins were correlated with stallion fertility utilizing regression analysis. Fertility was quantified by assigning a breeding score for each stallion. Each score was calculated by dividing the number of conceptions by the number of breedings for each stallion for four successive breeding seasons (1992-1995). Ejaculates from stallions of known ...
Kirsch K, Detilleux J, Serteyn D, Sandersen C.Portable blood gas analyzers are used to facilitate diagnosis and treatment of disorders related to disturbances of acid-base and electrolyte balance in the ambulatory care of equine patients. The aim of this study was to determine whether 2 portable analyzers produce results in agreement with a stationary analyzer. Blood samples from 23 horses hospitalized for various medical reasons were included in this prospective study. Blood gas analysis and electrolyte concentrations measured by the portable analyzers VetStat and epoc were compared to those produced by the cobas b 123 analyzer via conco...
Melnick JL, Schmidt NJ, Hampil B, Ho HH.This paper describes the preparation of seven combination pools of equine antisera, designated J though P, for identification of 19 coxsackievirus A immunotypes. Each pool is composed of 4 to 6 antisera; the serotypes included are A1-6, 8, 10-15, and 17-22. These pools, unlike the previously prepared A-H enterovirus pools, were lyophilized from volumes of 0.5 ml dispensed into 5-ml vials, and when rehydrated with 5 ml of diluent provide 50-antibody-unit material ready for use in identification tests without further dilution. Procedures for using the antiserum pools are given, and guidance is p...
De Santis T, Casavola V, Reshkin SJ, Guerra L, Ambruosi B, Fiandanese N, Dalbies-Tran R, Goudet G, Dell'Aquila ME.The extracellular calcium-sensing receptor (CASR) plays an important role in cells involved in calcium (Ca2+) homeostasis by directly sensing changes in the extracellular Ca2+ ion concentration. We previously reported the localization and quantitative expression of CASR protein in human oocytes. In this study, we examined the expression and the functional role of CASR during oocyte meiotic maturation in a large mammal animal model, the horse. As in humans, CASR protein was found to be expressed in equine oocytes and cumulus cells. Western-blot analysis revealed a single 130 kDa band in denuded...
Cheung HW, Wong KS, Lin VYC, Wan TSM, Ho ENM.The misuse of genetic manipulation technology to enhance athletic performance is termed gene doping which is prohibited in human sports, horseracing, and equestrian sports. Although many qPCR assays have been developed, most assays employ genomic DNA (gDNA) from humans, non-human primates, and mice as a background and they may not be applicable for testing horse samples. This study aimed to develop a qPCR assay for the detection of human erythropoietin (hEPO) transgene in horse blood cells where the viral vectors used in gene therapy can reside for months. For the detection of hEPO transgene, ...
Zhang W, Cui S, Cheng XL, Wei F, Ma S.In this study, probe/primers of high specificity and sensitivity were selected to analyze donkey-hide gelatin for donkey DNA and to look for horse, ox, and pig DNA as possible adulterants. The mitochondrial CO I genes in donkey, horse, and ox were selected as target sequences for design and synthesis of three pairs of specific probes and primers. In addition, eight pairs of probe/primers were obtained via literature search. Out of these eleven groups of probe/primers, those with the highest specificity and sensitivity were selected, which was fulfilled by the screening firstly with animal hide...
Surasi K, Cummings KJ, Hanson C, Morris MK, Salas M, Seftel D, Ortiz L, Thilakaratne R, Stainken C, Wadford DA.The Abbott BinaxNOW rapid antigen test is cheaper and faster than real-time reverse transcription PCR (rRT-PCR) for detecting severe acute respiratory syndrome coronavirus 2. We compared BinaxNOW with rRT-PCR in 769 paired specimens from 342 persons during a coronavirus disease outbreak among horse racetrack workers in California, USA. We found positive percent agreement was 43.3% (95% CI 34.6%-52.4%), negative percent agreement 100% (95% CI 99.4%-100%), positive predictive value 100% (95% CI 93.5%-100%), and negative predictive value 89.9% (95% CI 87.5%-92.0%). Among 127 rRT-PCR-positive ...
Milinovich GJ, Burrell PC, Pollitt CC, Bouvet A, Trott DJ.Four Gram-positive, catalase-negative, coccoid-shaped isolates were obtained from the caecum and rectum of horses with oligofructose-induced equine laminitis. Phenotypic and phylogenetic studies were performed on these isolates. Initial biochemical profiling assigned two of the isolates to Streptococcus bovis. The other two isolates, however, could not be assigned conclusively on the basis of their biochemical profiles. Gene sequence analysis demonstrated that the four new isolates were related most closely to Streptococcus suis based on the 16S rRNA gene and to Streptococcus orisratti based o...
Chapman MR, Kearney MT, Klei TR.Historically, surveys of equine parasites either are not quantitative in regard to prevalence and intensities of cyathostome species, or if quantitative, are estimates based on the identification of a very small sample of the population. Commonly 100-200 worms are identified. In the current study cyathostomes from 10 ponies were counted and identified to species in subsets of approximately 200 worms each from 5% aliquots of the large intestine contents until all worms in the aliquot were examined. A mean of 10.9+/-4.3 species were identified by examining 200 cyathostomes from each animal. This...
Stewart JM, Blakely JA, Johnson MD.Non-covalent modification of cytochrome c may have implications for electron transport and energy metabolism. We examined the interaction of various fatty acids (FAs), their coenzyme A and carnitine esters, and fatty alcohols with horse heart ferrocytochrome c. A comparison of FAs indicated a minimum chain length of 14 carbons was required for significant effect on the ferroheme chromophore and major changes in electronic spectra. Coenzyme A and carnitine esters interacted less strongly than FAs whereas long-chain alcohols did not interact with the protein. We found a single, saturable FA bind...
Gornik K, Moore P, Figueiredo M, Vandenplas M.Human corneal cells have detectable levels of TLRs 1-10. TLRs 2 and 4 are the major corneal receptors, recognizing the PAMPs associated with fungal invasion in humans. The conjunctiva and limbus contain TLRs 2, 4, and 9. Our purpose was to determine the expression of TLRs 2, 3, 4, 6, 9, and MD-2 in the normal equine cornea, conjunctiva, and limbus. Methods: Corneal, limbal, and conjunctival tissues were collected from seven euthanized horses having no evidence of ocular disease. RNA extraction with DNase-1 digestion was performed followed by RT-PCR to determine expression of TLRs 2, 3, 4, 6, 9...
Voelter-Ratson K, Monod M, Unger L, Spiess BM, Pot SA.To characterize the conjunctival fungal flora and to determine the susceptibility of 2 isolated molds to antifungal drugs in samples of 64 healthy horses from The National Stud in Switzerland. Methods: Conjunctival cytobrush samples were collected from both eyes of 64 ophthalmologically normal horses in August 2012 and subsequently cultured on Sabouraud's agar medium. Growing fungi were identified and counted. Etests or broth microdilution tests for Aspergillus fumigatus and Eurotium amstelodami were carried out to determine antifungal drug sensitivity. These species had previously been detect...
Langemeier JL, Cook SJ, Cook RF, Rushlow KE, Montelaro RC, Issel CJ.Control of equine infectious anemia (EIA) is currently based on detection of anti-EIA virus (EIAV) antibodies. However, serologic diagnostic methods may give false-negative results in infected horses that fail to respond adequately or are in the early stages of infection. We developed a reverse transcriptase nested PCR (RT-nPCR) assay for the detection of viral gag gene sequences in plasma from EIAV-infected horses. The ability of RT-nPCR to detect field strains of EIAV was investigated by assaying plasma samples from 71 horses stabled on EIA quarantine ranches. Positive PCR signals were detec...
Gross MK, Toscano DG, Toscano WA.Calmodulin (CaM), the calcium binding protein that modulates the activity of a number of key regulatory enzymes, is present at high levels in sperm. To determine whether CaM regulates adenylate cyclase in mammalian sperm, the actions of EGTA and selected CaM antagonists on a solubilized adenylate cyclase from mature equine sperm were examined. The activity of equine sperm adenylate cyclase was inhibited by EGTA in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of 2 mM. Equine sperm adenylate cyclase was also inhibited in a concentration-dependent manner by...
Zientara S, Sailleau C, Moulay S, Cruciere C.A single tube reverse transcription-polymerase chain reaction (RT-PCR) method for detection of African horse sickness virus (AHSV) in splenic tissues from infected horses is described. Double stranded RNA was extracted from infected organs of horses and used to produce complementary DNA (cDNA) with the two primers selected for the PCR. The 1179 bp amplified product (the segment 7 which encodes for VP 7), detected by electrophoresis on agarose gel and ethidium bromide staining, was hydrolysed with eight restriction endonucleases for characterization of the AHSV. The sensitivity of this method i...
Miglio A, Morelli C, Maresca C, Felici A, Di Gianbattista A, Antognoni MT.Serum protein electrophoresis (SPE) reference intervals (RIs) have been evaluated in different horses, but no specific values are shown for equine breeds as previously described in other species (dogs, cats), and no studies have been performed on SPE in draft horses. Objective: This study aimed to determine RIs for SPE in heavy draft horses (Italian Heavy Draft Horse-IHDH) living in central Italy. A comparison between different physiologic states (pregnancy and no pregnancy) and ages (foals and adults) was executed. Methods: Blood samples were collected from 215 apparently healthy horses (mare...
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...
Torgeman A, Mador N, Dorozko M, Lifshitz A, Eschar N, White MD, Wolf DG, Epstein E.Assuring viral safety of horse plasma-derived products is fundamental for ethical and regulatory reasons. We previously demonstrated the ability of pepsin digestion at low pH to inactivate West Nile and Sindbis viruses in horse plasma. The present study further examined the efficiency of pepsin digestion to inactivate four additional viruses: HSV-1 and BVDV (lipid-enveloped), BPV and Reo-3 (nonenveloped). These viruses were spiked into hyperimmunized horse plasma against botulinum toxin and subjected to low pH (3.2) alone or combined with pepsin digestion (1200 units/ml). Peptic digestion inac...
Sato F, Kanno T, Nagasawa S, Yanaihara N, Ishida N, Hasegawa T, Iwanaga T.We investigated the existence of chromogranin A (CgA) in salivary glands of the horse by Western blotting and enzyme immunoassay (EIA) using an antiserum against a peptide sequence of equine CgA. We also compared its cellular distribution between the horse and rat salivary glands with a tyramide signal amplification immunofluorescence technique. Western blotting gave three significant immunoreactive bands (74, 56 and 48 kDa) in adrenal medulla and three major salivary glands of horses. Immunoreactivities for CgA measured by EIA in horses were 154.05 +/- 41.46, 20.32 +/- 5.59 and 4.43 +/- 2.23 ...
Pavulraj S, Bera BC, Joshi A, Anand T, Virmani M, Vaid RK, Shanmugasundaram K, Gulati BR, Rajukumar K, Singh R, Misri J, Singh RK, Tripathi BN....Equine influenza viruses (EIV)-H3N8 continue to circulate in equine population throughout the world. They evolve by the process of antigenic drift that leads to substantial change in the antigenicity of the virus, thereby necessitating substitution of virus strain in the vaccines. This requires frequent testing of the new vaccines in the in vivo system; however, lack of an appropriate laboratory animal challenge model for testing protective efficacy of equine influenza vaccine candidates hinders the screening of new vaccines and other therapeutic approaches. In the present investigation, BALB/...
Wills JM, Watson G, Lusher M, Mair TS, Wood D, Richmond SJ.This paper describes the isolation and characterisation of a strain of Chlamydia psittaci obtained from a nasal swab taken from a horse with serous nasal discharge. Initial isolation was achieved in cycloheximide-treated McCoy cell monolayers. Chlamydial inclusions stained by immunofluorescence either with a rabbit antiserum raised against C. psittaci or with a monoclonal antibody directed against the genus-specific lipopolysaccharide antigen were single and compact. They did not stain with iodine or with a monoclonal antibody reactive against Chlamydia trachomatis. The agent was re-isolated i...
Dong JB, Zhu W, Cook FR, Goto Y, Horii Y, Haga T.Equine infectious anemia (EIA) has posed a major challenge and caused significant losses to the equine industry worldwide. PCR detection methods have considerable potential as an adjunct to conventional serological diagnostic techniques. However, most published PCR methods, including that recommended by the OIE, were designed using laboratory-adapted virus strains and do not function with field isolates of EIA virus (EIAV). In the present study, a nested PCR assay for detection of EIAV proviral DNA in peripheral blood cells of naturally infected horses was developed. Primer sets were designed ...
Raeside JI, Christie HL, Renaud RL, Waelchli RO, Betteridge KJ.Because estradiol (E(2)) production by the early equine conceptus is considered crucial to the establishment of pregnancy, the amounts of E(2), estrone (E(1)), and their sulfates (E(2)S, E(1)S) were measured by RIA in yolk-sac fluid of 63 conceptuses collected by transcervical lavage over the period of 11-26 days after ovulation. Amounts increased significantly with age of conceptus, especially for E(1)S. Then, the metabolism of E(2), which may be highly relevant for its action, was examined in the conceptus and endometrium over the period when the conceptus ceases to migrate within the uterus...
Love CC, Brinsko SP, Rigby SL, Thompson JA, Blanchard TL, Varner DD.The relationship between seminal plasma level (0, 10, or 20%) and extender type [Kenney type (EZ-Mixin-CST) or Kenney-modified Tyrodes-KMT] to the susceptibility of sperm DNA to denaturation and sperm motility measures were investigated in cooled (5 degrees C) stallion sperm. Three ejaculates from each of three fertile stallions were collected in an artificial vagina and processed as follows: diluted one part uncentrifuged semen with four parts of extender to a final concentration of 20% seminal plasma in either CST or KMT (20% CST; 20% KMT); diluted to a final concentration of 25 million sper...
Renzi S, Lombardo T, Dotti S, Dessì SS, De Blasio P, Ferrari M.The advent of stem cells and stem cell-based therapies for specific diseases requires particular knowledge of laboratory procedures, which not only guarantee the continuous production of cells, but also provide them an identity and integrity as close as possible to their origin. Their cryopreservation at temperatures below -80°C and typically below -140°C is of paramount importance. This target can be achieved by incorporating high molar concentrations of cryoprotectant mixtures that preserve cells from deleterious ice crystal formation. Usually, dimethyl sulfoxide (DMSO) and animal proteins...
Connard SS, Linardi RL, Even KM, Berglund AK, Schnabel LV, Ortved KF.The immunomodulatory properties of mesenchymal stem cells (MSCs) have been studied extensively due to their increasing clinical application for tissue regeneration and repair following culture expansion. We have studied the effect of continuous passage on the immunomodulatory capacity of equine bone marrow-derived MSCs (BM-MSCs). Equine BM-MSCs were isolated and culture expanded to passage three, six, and nine (P3, P6, P9). Immunomodulatory properties of each passage were assessed using a T cell proliferation assay and cytokine synthesis following stimulation with interferon gamma (IFN-γ). Re...
