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.
Chen JW, Uboh CE, Robinson MA, Jiang Z, Soma LR.To evaluate plasma interleukin 6 (IL-6) concentration in Standardbred racehorses by means of a novel ELISA following validation of the assay for use with equine plasma samples. Methods: Plasma samples obtained from 25 Thoroughbreds for use in assay validation and from 319 Standardbred racehorses at rest 2 to 2.5 hours prior to warm-up and racing. Methods: A sandwich ELISA was developed with equine anti-IL-6 polyclonal antibody and the biotin-streptavidin chemical interaction to enhance sensitivity. The assay was validated for specificity, sensitivity, precision, and accuracy by use of both rec...
Rose BV, Cabrera-Sharp V, Firth MJ, Barrelet FE, Bate S, Cameron IJ, Crabtree JR, Crowhurst J, McGladdery AJ, Neal H, Pynn J, Pynn OD, Smith C....Early pregnancy loss occurs in 6-10% of equine pregnancies making it the main cause of reproductive wastage. Despite this, reasons for the losses are known in only 16% of cases. Lack of viable conceptus material has inhibited investigations of many potential genetic and pathological causes. We present a method for isolating and culturing placental cells from failed early equine pregnancies. Trophoblast cells from 18/30 (60%) failed equine pregnancies of gestational ages 14-65 days were successfully cultured in three different media, with the greatest growth achieved for cells cultured in Amnio...
Boyle AG, Rankin SC, D￮ L, Boston RC, Wheeler-Aceto H.Bacterial culture and polymerase chain reaction (PCR) assays for the detection of Streptococcus equi in nasopharyngeal washes (NPW) and guttural pouch lavage (GPL) samples have low sensitivity. In human diagnostics, processing of samples with flocked swabs has improved recovery rates of bacterial agents because of improved surface area and elution factors. Objective: For S. equi subsp. equi (S. equi) detection in NPW and GPL samples we hypothesized that: direct-PCR would be more reliable than flocked swab culture (FS culture); flocked swab PCR (FS-PCR) would be equivalent to direct-PCR; and FS...
Bełżecki G, Miltko R, Michałowski T, McEwan NR.This paper describes cultivation methods for ciliates from the digestive tract of horses. Members of three different genera were successfully grown in vitro for short periods of time. However, only cells belonging to the genus Blepharocorys, which resides in the horse's large intestine, were maintained for longer periods. This Blepharocorys culture was successfully grown in vitro after inoculation of freshly excreted horse faeces in culture medium containing a population of bacteria. The ciliates survived for over six months, and the density of their population varied between 1.7 × 10(3) and ...
Ellerbrock R, Canisso I, Feijo L, Lima F, Shipley C, Kline K.Urospermia is known to affect semen quality in many mammals, including stallions. Determinations of semen pH and creatinine and urea concentrations have been used to diagnose urine contamination in raw stallion semen. Unfortunately, practitioners suspecting urine contamination in cooled-shipped samples have no proven means to confirm the presence of urine. Therefore, the objectives of this study were (1) to assess the effects of urine contamination on sperm motility of extended fresh and cooled-stored stallion semen, (2) to evaluate the usefulness of semen color, odor, pH, and creatinine and u...
Svala E, Jin C, Rüetschi U, Ekman S, Lindahl A, Karlsson NG, Skiöldebrand E.The glycoprotein lubricin contributes to the boundary lubrication of the articular cartilage surface. The early events of osteoarthritis involve the superficial layer where lubricin is synthesised. Objective: To characterise the glycosylation profile of lubricin in synovial fluid from horses with osteoarthritis and study secretion and degradation of lubricin in an in vitro inflammation cartilage model. Methods: In vitro study. Methods: Synovial fluid samples collected from horses with joints with normal articular cartilage and structural osteoarthritic lesions; with and without osteochondral f...
Romão FT, Pereira PF, Flaiban KK, Dearo AC, Fernandes TM, Lisbôa JA.Treatment of metabolic acidosis using sodium bicarbonate solutions is safe when blood gas analysis is available. The evidence that solutions containing metabolisable buffers can be used as an alternative for treatment of metabolic acidosis in horses is of practical interest. Objective: To investigate the safety and efficacy of a polyionic solution containing 84 mEq/l of lactate (L84) for the correction of induced hyperchloraemic metabolic acidosis. Methods: Non-randomised crossover design. Methods: Five healthy, adult, crossbred horses were used. A solution containing 100 mmol/l of HCl was inf...
Stefańska I, Witkowski L, Rzewuska M, Dzieciątkowski T.Rhodococcus equi is the causative agent of rhodococcosis in horses, resulting in significant morbidity and mortality in foals. This bacterium has also been isolated from a variety of animals and is being increasingly reported as a cause of infection in humans, mainly in immunosuppressed individuals. Laboratory diagnostics of R. equi infections based only on conventional microbiological methods shows low accuracy and can lead to misidentification. The objective of the study was to develop and evaluate a real-time PCR assay for direct detection of R. equi in various clinical specimens, including...
Barton MH, Darden JE, Clifton S, Vandenplas M.To validate primer sets for use in reverse transcription quantitative PCR assays to measure gene expression of cytosolic phospholipase A2 (cPLA2) and microsomal prostaglandin E2 synthase 1 (mPGES1) in equine mononuclear cells and determine the effects of firocoxib, a selective cyclooxygenase 2 (COX-2) inhibitor, on COX-2, cPLA2, and mPGES1 gene expression following incubation of mononuclear cells with lipopolysaccharide (LPS). Methods: 8 healthy adult horses. Methods: Peripheral blood mononuclear cells were isolated by density gradient centrifugation and incubated at 37°C with medium alone, f...
Van Der Vekens N, Hunter I, Timm A, Decloedt A, De Clercq D, Deprez P, Goetze JP, van Loon G.Equine atrial natriuretic peptide (ANP) plasma concentrations are correlated with left atrial size. However, species-specific assays are lacking and the results from human assays are poorly reproducible. A new methodology called processing independent analysis (PIA) that measures the total proANP product in plasma has proven to be successful in human medicine, but has never been used in horses. The aims were to establish an equine proANP reference interval by measurement of the total proANP product using PIA and to examine the proANP concentrations in horses with atrial dilatation. Sample stab...
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/...
Gao W, Chan Y, You M, Lacap-Bugler DC, Leung WK, Watt RM.This study explored the range of bacterial taxa present within healthy subgingival (below the gum-line) niches in the horse oral cavity using 16S rRNA gene amplicon pyrosequencing. Pooled subgingival plaque samples were collected from approximately 200 sulcus sites from two horses (EQ1, EQ2) for analysis. A total of 14,260 quality-filtered pyrosequencing reads were obtained, which were assigned to 3875 operational taxonomic units (OTUs; 99% identity cut-off); 1907 OTUs for EQ1 and 2156 OTUs for EQ2. Diverse taxa from 12 phyla were identified, including Actinobacteria (3.17%), Bacteroidetes (25...
Claes A, Ball BA, Troedsson MH, Curry TE, Squires EL, Scoggin KE.The wide variation in circulating anti-Müllerian hormone (AMH) concentrations between mares is attributed to differences in antral follicle count (AFC) which may reflect follicular function. There are few data regarding variations in AFC and associated regulatory factors for AMH in the equine follicle during follicular development. Objective: To examine molecular and hormonal differences in the equine follicle in relation to variations in AFC and circulating AMH concentrations during follicular development and to identify genes co-expressed with AMH in the equine follicle. Methods: Observatio...
Postel A, Cavalleri JM, Pfaender S, Walter S, Steinmann E, Fischer N, Feige K, Haas L, Becher P.Novel viruses belonging to the genera Hepacivirus and Pegivirus have recently been discovered in horses and other animal species. Viral genomes of non-primate hepaciviruses (NPHV), equine pegivirus 1 (EPgV 1) and Theiler's disease associated virus (TDAV) were detected in a horse serum routinely used for cell culture propagation in our laboratory. Therefore, a study was carried out to further investigate the presence of these human Hepatitis C virus (HCV) related viruses in equine serum based products used in veterinary medicine and for research and to characterize the viral genomes. Without ex...
Conway ED, Stiles J, Townsend WM, Weng HY.OBJECTIVE To evaluate species differences and effects of storage duration and temperature on the anticollagenase efficacy of canine, feline, and equine serum on in vitro corneal degradation. SAMPLES Corneas and serum from dogs, cats, and horses. PROCEDURES Clinically normal corneas from dogs, cats, and horses were harvested within 2 hours after euthanasia. Serum samples from dogs, cats, and horses were collected and pooled by species. Corneal specimens were incubated with collagenase derived from Clostridium histolyticum, 5mM calcium chloride in saline (0.9% NaCl) solution, and feline, canine,...
Schellenbacher C, Shafti-Keramat S, Huber B, Fink D, Brandt S, Kirnbauer R.The consistent and specific presence of Equus caballus papillomavirus type 2 (EcPV2) DNA and mRNA in equine genital squamous cell carcinoma (gSCC) is suggestive of an etiological role in tumor development. To further validate this concept, EcPV2-neutralizing serum antibody titers were determined by an EcPV2 pseudovirion (PsV) neutralization assay. Furthermore, an EcPV2 L1 virus-like particle (VLP)-based vaccine was generated and its prophylactic efficacy evaluated in vivo. All 6/6 gSCC-affected, but only 3/20 tumor-free age-matched animals revealed EcPV2-neutralizing serum antibody titers by P...
Brudvig JM, Swenson CL.Rapid and precise measurement of total and differential nucleated cell counts is a crucial diagnostic component of cavitary and synovial fluid analyses. Objective: The objectives of this study included (1) evaluation of reliability and precision of canine and equine fluid total nucleated cell count (TNCC) determined by the benchtop Abaxis VetScan HM5, in comparison with the automated reference instruments ADVIA 120 and the scil Vet abc, respectively, and (2) comparison of automated with manual canine differential nucleated cell counts. Methods: The TNCC and differential counts in canine pleura...
González-Fernández L, Macedo S, Lopes JS, Rocha A, Macías-García B.Equine in vitro fertilization (IVF) is still inconsistent. In the present work, we studied how modified Whitten's (MW) medium and Tissue Culture Medium 199 (TCM) added with Foetal Bovine Serum (FBS; 10% v/v) or Bovine Serum Albumin (BSA; 7 mg/ml) affected equine gametes to subsequently run IVF trials. Compact (Cp) and expanded (Ex) cumuli equine oocytes were matured and placed in TCM or MW supplemented with BSA or FBS for 18-20 h (no sperm added). In Ex oocytes, TCM-199 added with FBS or BSA resulted in higher metaphase II (MII) rates (75.7% and 62.7%, respectively) than MW added with BSA (54%...
Bonelli F, Meucci V, Divers TJ, Jose-Cunilleras E, Corazza M, Tognetti R, Guidi G, Intorre L, Sgorbini M.The diseases most frequent associated with SIRS in adult horses are those involving the gastrointestinal tract. An early diagnosis should be the goal in the management of horses with SIRS. Objective: The objective of this study was to evaluate the plasma procalcitonin (PCT) concentration in healthy and SIRS horses to assess differences between the two groups. Methods: Seventy-eight horses (30 healthy and 48 SIRS). Methods: Prospective in vivo multicentric study. Horses were classified as SIRS if at least 2 of the following criteria were met: abnormal leukocyte count or distribution, hypertherm...
Turło A, Cywińska A, Czopowicz M, Witkowski L, Niedźwiedź A, Słowikowska M, Borowicz H, Jaśkiewicz A, Winnicka A.Training-induced muscle, skeletal and joint trauma may result in acute phase response reflected by the changes in the blood concentration of serum amyloid A (SAA) in racehorses. It remains yet unclear if such systemic reaction could be triggered by sport injuries and what is the impact of different types of musculoskeletal trauma on SAA concentrations in racehorses. This study aimed to determine changes in the SAA blood concentration in racehorses with different types of injuries of musculoskeletal system. Methods: The study involved 28 racehorses diagnosed after the race with bone fractures (...
Chung CJ, Grimm AL, Wilson CL, Balasuriya UB, Chung G, Timoney PJ, Bandaranayaka-Mudiyanselage CB, Lee SS, McGuire TC.In an effort to improve a competitive blocking enzyme-linked immunosorbent assay (cELISA) for antibody detection to Equine arteritis virus (EAV), antigen purified by anion-exchange membrane chromatography capsule (AEC) was evaluated. Virus purification by the AEC method was rapid and easily scalable. A comparison was made between virus purified by the AEC method with that obtained by differential centrifugation based on the following: 1) the relative purity and quality of EAV glycoprotein 5 (GP5) containing the epitope defined by monoclonal antibody 17B7, and 2) the relative sensitivity of a c...
Stefaniuk M, Ropka-Molik K.RNA sequencing (RNA-seq) by next-generation technology is a powerful tool which creates new possibilities in whole-transcriptome analysis. In recent years, with the use of the RNA-seq method, several studies expanded transcriptional gene profiles to understand interactions between genotype and phenotype, supremely contributing to the field of equine biology. To date, in horses, massive parallel sequencing of cDNA has been successfully used to identify and quantify mRNA levels in several normal tissues, as well as to annotate genes. Moreover, the RNA-seq method has been applied to identify the ...
Brinkmann J, Koudelka T, Keppler JK, Tholey A, Schwarz K, Thaller G, Tetens J.The production and consumption of mare's milk in Europe has gained importance, mainly based on positive health effects and a lower allergenic potential as compared to cows' milk. The allergenicity of milk is to a certain extent affected by different genetic variants. In classical dairy species, much research has been conducted into the genetic variability of milk proteins, but the knowledge in horses is scarce. Here, we characterize two major forms of equine αS2-casein arising from genomic 1.3 kb in-frame deletion involving two coding exons, one of which represents an equid specific duplicati...
Cieslak J, Mackowski M, Czyzak-Runowska G, Wojtowski J, Puppel K, Kuczynska B, Pawlak P.Apart from the well-known role of somatic cell count as a parameter reflecting the inflammatory status of the mammary gland, the composition of cells isolated from milk is considered as a valuable material for gene expression studies in mammals. Due to its unique composition, in recent years an increasing interest in mare's milk consumption has been observed. Thus, investigating the genetic background of horse's milk variability presents and interesting study model. Relying on 39 milk samples collected from mares representing three breeds (Polish Primitive Horse, Polish Cold-blooded Horse, Pol...
Holl HM, Gao S, Fei Z, Andrews C, Brooks SA.Laminitis, the structural failure of interdigitated tissue that suspends the distal skeleton within the hoof capsule, is a devastating disease that is the second leading cause of both lameness and euthanasia in the horse. Current transcriptomic research focuses on the expression of known genes. However, as this tissue is quite unique and equine gene annotation is largely derived from computational predictions, there are likely yet uncharacterized transcripts that may be involved in the etiology of laminitis. In order to create a novel annotation resource, we performed whole transcriptome seque...
Ghassab S, Dulin J, Bertone AL.To compare clotting efficiency of platelet-rich plasma (PRP) and concentrated platelet-poor plasma (cPPP) to citrated whole blood after activation by autologous thrombin, bovine thrombin, or calcium chloride (CaCl2 ). Methods: Experimental study. Methods: Healthy adult horses (n = 6). Methods: PRP and cPPP were prepared by commercial devices. Using thromboelastography, clotting variables were compared after activation of citrated autologous blood, PRP, and cPPP by autologous thrombin, bovine thrombin, or CaCl2 , respectively. Results: PRP had the greatest clot strength and quickest clot rate, ...
Bannai H, Nemoto M, Tsujimura K, Yamanaka T, Maeda K, Kondo T.To increase the sensitivity of an enzyme-linked immunosorbent assay (ELISA) for equine herpesvirus type 4 (EHV-4) that uses a 12-mer peptide of glycoprotein G (gG4-12-mer: MKNNPIYSEGSL) [4], we used a longer peptide consisting of a 24-mer repeat sequence (gG4-24-mer: MKNNPIYSEGSLMLNVQHDDSIHT) as an antigen. Sera of horses experimentally infected with EHV-4 reacted much more strongly to the gG4-24-mer peptide than to the gG4-12-mer peptide. We used peptide ELISAs to test paired sera from horses naturally infected with EHV-4 (n=40). gG4-24-mer ELISA detected 37 positive samples (92.5%), whereas ...
Ilomuanya M, Uboh C, Ciallella J, Li X, Liu Y, Ifudu N, Azubuike C, Igwilo C.Treatment of racehorses with bicarbonate solutions to manage acidosis and muscle cramps prior to competition is banned in Pennsylvania (PA). Use of excess bicarbonate in horses causes diarrhea, requiring treatment with an antibiotic such as metronidazole (MTNZ). At present no method exists for detecting MTNZ in equine plasma. Thus, a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the detection, quantification and confirmation of MTNZ was developed. Methods: The analyte was recovered from plasma by liquid-liquid extraction using methyl tert-butyl ether and separated on an ...
Sjostrom L, al-Abdulla IH, Rawat S, Smith DC, Landon J.Commercial antivenoms produced in horses were compared with monospecific antivenoms raised in sheep against Crotalus durissus terrificus, Crotalus atrox, Crotalus adamanteus, Micrurus fulvius fulvius, Naja naja, Naja kaouthia, Echis ocellatus, Vipera lebetina deserti, Vipera berus berus and Vipera ammodytes ammodytes venom. Antibodies raised by immunizing sheep with C. d. terrificus venom were more effective than their equine counterparts in preventing lethal toxicity in mice (ED50), in inhibiting the venom's pharmacological effects (haemolysis, platelet aggregation and coagulation), and in ne...
Martoriati A, Caillaud M, Goudet G, Gérard N.Interleukin 1 beta (IL-1 beta) inhibits the LH-induced resumption of meiosis of equine oocytes in vitro. The present study was performed to clarify this inhibitory effect of IL-1 beta by testing increasing concentrations of IL-1 beta, and by measuring the effect of addition of IL-1 receptor antagonist (IL-1RA) to the culture medium. The effect of IL-1 beta on epidermal growth factor (EGF)-induced resumption of meiosis was also studied. Cumulus-oocyte complexes (COCs) were collected from subordinate follicles on ovaries obtained from an abattoir. In five distinct experiments, COCs were cultured...
Bełżecki G, Miltko R, Michałowski T, McEwan NR.This paper describes cultivation methods for ciliates from the digestive tract of horses. Members of three different genera were successfully grown in vitro for short periods of time. However, only cells belonging to the genus Blepharocorys, which resides in the horse's large intestine, were maintained for longer periods. This Blepharocorys culture was successfully grown in vitro after inoculation of freshly excreted horse faeces in culture medium containing a population of bacteria. The ciliates survived for over six months, and the density of their population varied between 1.7 × 10(3) and ...
Epstein KL, Brainard BM, Lopes MA, Barton MH, Moore JN.To develop a standardized technique for thrombelastography (TEG) analysis in healthy adult horses, with and without the ex vivo addition of tissue factor (TF) as an activator. To determine reference intervals for TEG parameters in the horse, and to determine if traditional coagulation tests correlate with TEG. Methods: Prospective, observational. Methods: Veterinary teaching hospital. Methods: Twenty-six healthy adult horses. Methods: None. Results: Thrombelastography with (TF-TEG) and without (TEG) the addition of TF performed by 4 operators. Coagulation profiles (prothrombin time, activated ...
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...
Sweeney CR, Russell GE.To examine total protein concentration and cell counts of sequentially collected samples of CSF to determine whether blood contamination decreases in subsequent samples and whether formulas used to correct nucleated cell count and total protein concentration are accurate. Methods: Case series. Methods: 22 horses. Methods: For each horse, 3 or 4 sequential 2-ml samples of CSF were collected from the subarachnoid space in the lumbosacral region into separate syringes, and blood was obtained from the jugular vein. Total protein concentration, nucleated cell count, and RBC counts were determined i...
Marth CD, Firestone SM, Hanlon D, Glenton LY, Browning GF, Young ND, Krekeler N.Persistent mating-induced endometritis (PMIE) severely decreases fertility in horses. The aim of the present study was to evaluate differences between horses susceptible to PMIE and a control group in terms of the expression of selected immune response and effector genes, and the effects of oestrous cycle stage on this expression. Endometrial biopsies from 18 uterine samples of mares in the control group (eight in dioestrus, 10 in oestrus) and 16 PMIE-susceptible mares (four in dioestrus, 12 in oestrus) were analysed by quantitative real-time reverse transcription-polymerase chain reaction. Ge...
Dunnett M, Harris RC, Orme CE.A simple, robust and reproducible analytical method for the determination of phosphocreatine (PCr), creatine (Cr) and creatinine (Cn) in equine skeletal muscle is presented. The technique used isocratic reverse-phase ion-pairing high-performance liquid chromatography. Neutralized perchloric acid extracts of equine muscle biopsies were analysed and the values obtained were compared with determinations from an established enzymic procedure. Good resolution of all three metabolites was achieved within a retention time of less than 11 min. Linearity for each metabolite within the concentration ran...
Bramer SA, Macedo A, Klein C.Secretion of histotroph during the prolonged pre-implantation phase in mares is crucial to pregnancy maintenance, manifested as increased embryonic loss in mares with age-related endometrial degeneration. Glycogen content of uterine histotroph is higher during the progesterone-dominated phase of the estrous cycle in mares, but regulatory mechanisms are not well understood. Methods: mRNA expression of glycogen-metabolizing enzymes (HK1, HK2, GSK3B, GYS1, PEPCK, PKM, PYGM) in endometrial samples were compared among mares in anestrus, estrus, and at Day 12 of diestrus and pregnancy. In addition, ...
Lavoie JP, Lefebvre-Lavoie J, Leclere M, Lavoie-Lamoureux A, Chamberland A, Laprise C, Lussier J.Gene expression analyses are used to investigate signaling pathways involved in diseases. In asthma, they have been primarily derived from the analysis of bronchial biopsies harvested from mild to moderate asthmatic subjects and controls. Due to ethical considerations, there is currently limited information on the transcriptome profile of the peripheral lung tissues in asthma. Objective: To identify genes contributing to chronic inflammation and remodeling in the peripheral lung tissue of horses with heaves, a naturally occurring asthma-like condition. Methods: Eleven adult horses (6 heaves-af...
González Medina S, Hyde C, Lovera I, Piercy RJ.Hypoglycin A (HGA) toxicity, following ingestion of material from certain plants, is linked to an acquired multiple acyl-CoA dehydrogenase deficiency known as atypical myopathy, a commonly fatal form of equine rhabdomyolysis seen worldwide. Whilst some plants are known to contain this toxin, little is known about its function or the mechanisms that lead to varied HGA concentrations between plants. Consequently, reliable tools to detect this amino acid in plant samples are needed. Analytical methods for HGA detection have previously been validated for the food industry, however, these technique...
Marsh AE, Barr BC, Madigan J, Lakritz J, Conrad PA.To identify Sarcocystis neurona-specific DNA sequences in the nuclear small subunit ribosomal RNA (nss-rRNA) gene that could be used to distinguish S neurona from other closely related protozoal parasites, and to evaluate a polymerase chain reaction (PCR) test, using broad based primers and a unique species-specific probe on CSF for detection of S neurona in equids. Methods: Sequencing of the nuclear small subunit ribosomal RNA gene from a new S neurona isolate (UCD 1) was performed. The sequence was compared with that of other closely related Sarcocystidae parasites. From this sequence, conse...
Choi YH, Roasa LM, Love CC, Varner DD, Brinsko SP, Hinrichs K.This study was conducted to evaluate in vivo and in vitro development of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection. Oocytes were collected from slaughterhouse-derived ovaries, matured in vitro, and injected with frozen-thawed stallion sperm. In vivo development was assessed after transfer of injected oocytes to the oviducts of recipient mares. Mares were killed 7.5-8.5 days after transfer and the uterus and oviducts flushed for embryo recovery. Of 132 injected oocytes transferred, 69 (52%) were recovered; of these, 25 (36%) were blastocysts with a blastocoe...
Nemoto M, Hata H, Higuchi T, Imagawa H, Yamanaka T, Niwa H, Bannai H, Tsujimura K, Kondo T, Matsumura T.We evaluated antigen detection kits for human rotavirus with regard to their usefulness for diagnosing equine rotavirus infection. Limiting dilution analyses showed that of the seven kits investigated the Dipstick `Eiken' Rota (Dipstick) had the highest sensitivity to two serotypes of equine rotavirus. The Dipstick did not cross-react with several equine intestinal pathogens. An investigation using 249 fecal samples indicated that the sensitivity of the Dipstick was 81.9% and 47.3%, and its specificity was 98.2% and 99.0%, and its concordance rate was 92.8% and 68.3%, compared with values obta...
Artiushin S, Tong Y, Timoney J, Lemieux B, Schlegel A, Kong H.A simple and portable assay for detection of Streptococcus equi subspecies equi has been developed based on amplification of S. equi-specific sequence using a thermophilic helicase-dependent reaction followed by visual detection of the amplicon in a disposable lateral flow cassette. An experimental kit (IsoAmp™ SE) was evaluated. Analytical sensitivity was 50 copies of S. equi genomic DNA per reaction. The IsoAmp SE assay had 100% specificity when applied to nasal swabs and washes. The assay was more sensitive than culture but less sensitive than nested polymerase chain reaction (PCR). The t...
Owens SD, Snipes J, Magdesian KG, Christopher MM.Blood typing before transfusion minimizes the risk of transfusion reactions and prevents immunization of the recipient against incompatible RBC antigens. The major RBC antigens that warrant identification before packed RBC or whole blood transfusions in horses are Ca and Aa. Standard blood-typing protocols are time-consuming (2.5-3.0 hours) and impractical in emergency settings. Objective: The purpose of this study was to determine whether equine RBCs could be typed for Ca and Aa antigens using sera from horses with RBC antibodies in a modified rapid (15 minute) blood-typing protocol. Methods:...
Agius CT, Nagesha HS, Studdert MJ.A new equine herpesvirus, provisionally designated equine herpesvirus 5 (EHV5; Browning and Studdert (1987) J. Gen. Virol. 68, 1441-1447), was examined for the degree of genomic difference from equine herpesvirus 2 (EHV2) by Southern hybridizations. EHV5 and EHV2 whole genomic DNA probes were highly specific for homologous DNA only, indicating that significant genomic difference exists between the two viruses. Restriction endonuclease analysis of EHV5 strain 2-141 (EHV5.2-141) revealed that the genome is 179 kb and exists as a single isomer. Clones representing 82% of the genome were obtained ...
Leeb T, Müller EJ, Roosje P, Welle M.Molecular genetics has made significant advances in the analysis of hereditary dermatoses during the last several years. Objective: To provide an update on currently available genetic tests for skin diseases of dogs, cats and horses, and to aid the veterinary clinician in the appropriate selection and applications of genetic tests. Methods: The scientific literature on the topic was critically reviewed. The list of known causative variants for genodermatoses and hair morphology traits was compiled by searching the Online Mendelian Inheritance in Animals (OMIA) database. Results: Genetic testin...
Mansfield LS, Schott HC, Murphy AJ, Rossano MG, Tanhauser SM, Patterson JS, Nelson K, Ewart SL, Marteniuk JV, Bowman DD, Kaneene JB.Sarcocystis neurona is a protozoan parasite that can cause neurological deficits in infected horses. The route of transmission is by fecal-oral transfer of sporocysts from opossums. However, the species identity and the lifecycle are not completely known. In this study, Sarcocystis merozoites from eight isolates obtained from Michigan horses were compared to S. neurona from a California horse (UCD1), Sarcocystis from a grackle (Cornell), and five Sarcocystis isolates from feral opossums from Michigan. Comparisons were made using several techniques. SDS-PAGE analysis with silver staining showed...
Grippa E, Santini L, Castellano G, Gatto MT, Leone MG, Saso L.Ethyl acetate extracts of equine serum, containing 0-5 microg/ml of hydrocortisone (HYD), dexamethasone (DEX), oxyphenbutazone (OPB), indomethacin (IND), phenylbutazone (PB) and probenecid as internal standard, were evaporated with nitrogen, resuspended in methanol and analyzed by HPLC, using a C-18 column equilibrated with 51:49 acetonitrile-water, 0.1% trifluoroacetic acid, at 1 ml/min. The eluate was monitored at 254 nm. The selectivity (inter-assay C.V.<4%), sensitivity (limits of quantitation of 0.25 microg/ml for HYD, DEX and IND, 0.5 microg/ml for PB and 1 microg/ml for OPB, despite ...
Lonning SM, Zhang W, Leib SR, McGuire TC.Cytotoxic T lymphocytes (CTL) appear to be critical in resolving or reducing the severity of lentivirus infections. Retroviral vectors expressing the Gag/Pr or SU protein of the lentivirus equine infectious anemia virus (EIAV) were constructed and used to evaluate EIAV-specific CTL responses in horses. Three promoters, cytomegalovirus, simian virus SV40, and Moloney murine sarcoma virus (MoMSV) long terminal repeat (LTR), were used, and there was considerable variation in their ability to direct expression of Gag/Pr and SU. Vectors expressing EIAV proteins under the direction of MoMSV LTR and ...
Bannai H, Nemoto M, Tsujimura K, Yamanaka T, Kondo T, Matsumura T.Virus-neutralizing (VN) testing is essential for evaluating virus-specific immunity in equine herpesvirus type-1 (EHV-1) infection. We developed a focus-reduction neutralization test (FRNT) for EHV-1 using 96-well plates for faster large-scale testing with sufficient sensitivity. We used an overlay medium containing Avicel (FMC Biopolymer), a microcrystalline cellulose with lower viscosity than the methylcellulose. The foci were visualized by immuno-staining with anti-EHV-1 gp14 monoclonal antibody. The FRNT successfully detected seroconversion in horses experimentally infected with EHV-1 (n =...
Magnuson NS, Perryman LE, Wyatt CR, Mason PH, Talmadge JE.Peripheral blood mononuclear cells from foals with hereditary severe combined immunodeficiency (SCID) have morphologic characteristics of large granular lymphocytes (LGL). Attempts to demonstrate cytotoxic activity were without success unless the LGL were incubated with 100 U of human recombinant interleukin 2 (rIL 2)/ml for 24 hr. With rIL 2 incubation, low effector to target ratios (10:1) consistently yielded high levels of cytotoxic activity (30 to 50%) in a standard 4-hr 51Cr-release assay using YAC-1 lymphoma or K562 erythroleukemia cell lines as targets. Monoclonal antibody EqT12 reacted...
Henson FM, Davenport C, Butler L, Moran I, Shingleton WD, Jeffcott LB, Schofield PN.The effects of insulin and insulin-like growth factors (IGFs) I and II on fetal and foal chondrocytes were investigated in vitro. Chondrocytes from the lateral trochlear ridge of the distal femur were obtained from 2 fetuses (280 and 320 days gestation) and one 4-day-old foal and cultured. Membrane proteins consistent with type 1 and type 2 IGF receptors were demonstrated by radioligand cross linking and equilibrium binding analysis. It was demonstrated that both IGF-I and IGF-II acted as mitogens for isolated equine chondrocytes when present as the sole mitogenic factor in monolayer culture. ...
Pickles K, Pirie RS, Rhind S, Dixon PM, McGorum BC.Bronchoalveolar lavage fluid (BALF) samples are often subject to time delays, possibly with temperature fluctuations, between collection and processing. The aim of this study was to evaluate the effects of time, temperature and 2 different fixatives on equine BALF cytology, in order to develop guidelines for optimal equine BALF storage conditions. Total nucleated cell count (TCC), differential cell counts (DCC), absolute cell counts (ACC), cell viability, cell morphology and bacterial growth of BALF samples stored at 4, 18 (+/- addition of formalin- or alcohol-based fixatives) and 38 degrees C...
Sandbaumhüter FA, Thormann W.Glucuronidation catalyzed by uridine-5'-diphospho-glucuronosyl-transferases (UGTs) is the most important reaction in phase II metabolism of drugs and other compounds. O-glucuronidation is more common than N-glucuronidation. The anesthetic, analgesic and antidepressive drug ketamine is metabolized in phase I by cytochrome P450 enzymes to norketamine, hydroxynorketamine (HNK) diastereomers and dehydronorketamine (DHNK). Equine urine samples collected two hours after ketamine injection were treated with β-glucuronidase and analyzed with three enantioselective capillary electrophoresis assays. Co...
Fjeldsgaard BE, Paulsen BS.Extracts of horse dander (HD) and horse hair and skin scrapings (HHSS) have been compared with respect to their content of proteins and carbohydrates. The protein content of HD is more than double that of HHSS, while the carbohydrate content is of the same order. SDS-PAGE and IEF, both combined with immunoblotting, and CIE/CRIE showed the IgE-binding ability of the proteins/glycoproteins present in the two extracts. SDS-PAGE/immunoblotting showed the presence of mainly the same IgE-binding bands in the two extracts. Nine were detected in HD, and seven in HHSS. Four of these were glycoproteins....
Goudet G, Belin F, Bézard J, Gérard N.In the equine species, a large proportion of oocytes fail to complete meiosis during in-vitro culture. The biochemical and molecular basis of this failure is unknown. The meiotic cell cycle is controlled in part by the maturation-promoting factor (MPF) and the mitogen-activated protein kinase (MAPK). In this study, we evaluated the oocyte competence for in-vitro maturation and the expression of MPF components (p34cdc2 and cyclin B) and MAPK after in-vitro culture. The maturation rate was influenced by the culture medium and the physiological stage of the mare at the time of oocyte recovery. We...
Caron JP, Gandy JC, Schmidt M, Hauptman JG, Sordillo LM.To compare the effects of triamcinolone acetonide (TA) and methylprednisolone acetate (MPA) on expression of selected chondrocyte genes in recombinant equine interleukin-1β (reIL-1β) stimulated articular cartilage explants. Methods: In vitro experiment. Methods: Horses (n = 6). Methods: Articular cartilage explants from 2- to 3- year-old horses were exposed to reIL-1β in the presence and absence of TA and MPA at 10(-7) and 10(-6) M. Resting levels of mRNA of anabolic and catabolic genes of chondrocyte origin were quantified using qPCR after 6- and 12-hour incubations. Genes of interest incl...
Brown JJ, Thomson W, Clegg P, Eyre S, Kennedy LJ, Matthews J, Carter S, Ollier WE.The full extent of the polymorphism of ELA-DRA in Equidae is not yet known. Given the apparent differences in DRA polymorphisms between Equidae and other species, the aims of this study were to more fully characterize ELA-DRA, determine the extent of gene polymorphism and establish the allele-frequency distribution. An allele reference panel for the second exon of ELA-DRA was established by sequence-based typing of 69 equine DNA samples consisting of various breeds of domestic horse (Equus caballus), together with donkeys (Equus asinus), Grant's zebras (Equus boehmi) and one onager (Equus hemi...
