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Topic:Laboratory Methods
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.
Relative stiffness of 3 bandage/splint constructs for stabilization of equine midmetacarpal fractures. Determine the relative stiffness of 3 bandage/splint constructs intended for emergency fracture stabilization. Methods: Experimental model. A single plane free end deflection model was developed to simulate the forces placed on a bandage/splint construct during stabilization of a complete mid-metacarpal bone fracture. The total deflection of the model in one plane was measured following application of 3 different bandage/splint combinations including a classic, 3 layered Robert Jones Bandage (RJB) with a splint placed on the outside of the bandage (RJB-3), an RJB with splint placed after the f...
Opiorphin analysis in equine plasma and urine using hydrophilic interaction LC-MS. Due to opiorphin's analgesic and antidepressant functions, its illicit use is rumored in some racing jurisdictions. Opiorphin is very difficult to detect due to its hydrophilic nature and rapid degradation in plasma and urine samples. METHODOLOGY & RESULTS: We have developed a sensitive, reliable method for opiorphin detection and confirmation in equine samples, using EDTA to inhibit analyte degradation between the time of collection and analysis. Opiorphin was extracted by weak cation exchange followed by analysis using HILIC-MS/MS. The method was validated and the LOD was determined to b...
Effect of incubation temperature on the diagnostic sensitivity of the glanders complement fixation test. The complement fixation test (CFT) is the only serological test prescribed by the World Organisation for Animal Health (OIE) for the diagnosis of glanders in international trading of equids. However, false-positive reactions have caused financial losses to the animal owners in the past, and false-negative tests have resulted in the introduction of glanders into healthy equine populations in previously glanders-free areas. Both warm (incubation at 37°C for 1 h) and cold (overnight incubation at 4°C) procedures are recommended by the OIE for serodiagnosis of glanders. In a comparison of the se...
Anti-Müllerian hormone as an indicator of hemi-castrated unilateral cryptorchid horses. Anti-Müllerian hormone (AMH), a glycoprotein secreted from the fetal testis, is responsible for regression of the Müllerian duct in the male fetus. The aim of this study was to evaluate the usefulness of serum AMH as a biomarker for diagnosis of cryptorchidism in horses. Serum AMH concentrations were measured in intact stallions, hemi-castrated unilateral cryptorchid stallions, and geldings. In addition, expression of AMH was characterized in cryptorchid testes by immunohistochemistry. Serum AMH was detected in intact stallions (n=11, 13.3 ± 1.8 ng/ml) and in hemi-castrated cryptorchid stal...
Sarcocyst Development in Raccoons (Procyon lotor) Inoculated with Different Strains of Sarcocystis neurona Culture-Derived Merozoites. Sarcocystis neurona is considered the major etiologic agent of equine protozoal myeloencephalitis (EPM), a neurological disease in horses. Raccoon ( Procyon lotor ) is considered the most important intermediate host in the life cycle of S. neurona in the United States; S. neurona sarcocysts do mature in raccoon muscles, and raccoons also develop clinical signs simulating EPM. The focus of this study was to determine if sarcocysts would develop in raccoons experimentally inoculated with different host-derived strains of in vitro-cultivated S. neurona merozoites. Four raccoons were inoculated wi...
Chip-based nanoelectrospray ionization with Fourier transform mass spectrometric detection to screen for local anesthetics intended to mask limb sore in walking horses. We report a high-throughput chip-based nanoelectrospray ionization method coupled with Fourier transform mass spectrometry to screen for local anesthetics in samples collected by swabbing. These drugs have been used to mask pain on the limbs of walking horses after forbidden practices of soring or physical abuse. Optimized for lidocaine, the method afforded sub-ppm mass accuracy for nine local anesthetics included in the study. From doped cotton swabs, two third and all of the analytes were detected after adding 10 ng and 100 ng of each drug, respectively. Benzocaine and/or lidocaine were ...
Serum amyloid A, haptoglobin, and ferritin in horses with colic: Association with common clinicopathological variables and short-term outcome. Equine colic may be associated with an acute phase response (APR). Measurement of acute phase proteins (APPs) allows the detection of an APR and may help clinicians in monitoring the disease; however, the role of APPs in colic is unclear. This study aimed to evaluate the clinical usefulness of serum amyloid A (SAA), haptoglobin and ferritin in combination with an extended clinicopathological profile in equine colic. The medical records of 54 horses were retrospectively selected. Horses were grouped based on outcome (survivors vs. non-survivors), diagnosis (ischaemic/strangulating vs. non-ischa...
The transcriptome of equine peripheral blood mononuclear cells. Complete transcriptomic data at high resolution are available only for a few model organisms with medical importance. The gene structures of non-model organisms are mostly computationally predicted based on comparative genomics with other species. As a result, more than half of the horse gene models are known only by projection. Experimental data supporting these gene models are scarce. Moreover, most of the annotated equine genes are single-transcript genes. Utilizing RNA sequencing (RNA-seq) the experimental validation of predicted transcriptomes has become accessible at reasonable costs. To...
Using epidemics to map H3 equine influenza virus determinants of antigenicity. Equine influenza is a major cause of respiratory infections in horses and causes widespread epidemics, despite the availability of commercial vaccines. Antigenic drift within the haemagglutinin (HA) glycoprotein is thought to play a part in vaccination breakdown. Here, we carried out a detailed investigation of the 1989 UK outbreak, using reverse genetics and site-directed mutagenesis, to determine the individual contribution of amino acid substitutions within HA. Mutations at positions 159, 189 and 227 all altered antigenicity, as measured by haemagglutination-inhibition assays. We also compa...
Species-specific effects on non-enzymatic metmyoglobin reduction in vitro. Our objectives were to determine the non-enzymatic metmyoglobin reduction properties of bovine, porcine, and equine myoglobins and to characterize the effects of pre-incubation of 4-hydroxy-2-nonenal (HNE) with myoglobins on non-enzymatic metmyoglobin reduction in vitro. Purified bovine, porcine, and equine metmyoglobins (0.05 mM) were reduced at pH 5.6 and 7.4 in the presence or absence of HNE. Rates of metmyoglobin reduction were monitored by spectrophotometry, and myoglobin adducts were characterized by high-resolution mass-spectrometry. Results showed that the species origins of individual...
Influence of cryopreservation and mechanical stimulation on equine Autologous Conditioned Plasma (ACP®). To determine the influence of cryopreservation at two different temperatures on platelet concentration, growth factor (GF) levels and platelet activation parameters in equine ACP®; moreover, to determine if adding mechanical ACP® stimulation to freeze-thaw activation amplifies GF release from platelets. Methods: Firstly, blood from five horses was used to prepare ACP®. Platelet, platelet derived growth factor BB (PDGF-BB) and transforming growth factor β1 (TGF-β1) concentrations as well as mean platelet volume (MPV) and mean platelet component (MPC) were determined in fresh and correspond...
Accuracy of preimplantation genetic diagnosis in equine in vivo-recovered and in vitro-produced blastocysts. Preimplantation genetic diagnosis has great potential in the horse, but information on evaluation of equine embryo biopsy samples is limited. Blastocysts were biopsied using a Piezo drill and methods for whole-genome amplification (WGA) investigated. Results for 33 genetic loci were then compared between biopsy samples from in vitro-produced (IVP) and in vivo-recovered (VIV) blastocysts. Under the experimental conditions described, WGA using the Qiagen Repli-g Midi kit was more accurate than that using the Illustra Genomiphi V2 kit (98.2% vs 25.8%, respectively). Using WGA with the Qiagen kit,...
The role of sera from equine grass sickness on apoptosis induction in PC12 Tet-off p53 cell line. The pathogenesis of equine grass sickness (EGS) has not fully understood. A better understanding of the exact pathogenesis of diseases can help to make an accurate diagnosis. Previous studies reported some pathological damage of neuronal cells in EGS patients. In this study, primarily cytotoxicity of serum from three clinically EGS-diagnosed horses on PC12 Tet-off (PTO) cells was assessed. Subsequently, the apoptotic tests including cytochrome C release, caspase-3/7 activity measurement and DNA fragmentation assay were conducted to clarify the apoptotic effect of serum from EGS patients. Addit...
Effects of sodium citrate and acid citrate dextrose solutions on cell counts and growth factor release from equine pure-platelet rich plasma and pure-platelet rich gel. There is a lack information on the effects of the most commonly used anticoagulants for equine platelet rich plasmas (PRPs) elaboration on cell counts and growth factor release from platelet rich gels (PRGs). The aims of this study were 1) to compare the effects of the anticoagulants sodium citrate (SC), acid citrate dextrose solution A (ACD-A) and ACD-B on platelet (PLT), leukocyte (WBC) and on some parameters associated to platelet activation including mean platelet volume (MPV) and platelet distribution width (PDW) between whole blood, pure PRP (P-PRP) and platelet-poor plasma (PPP); 2) to ...
Nonstrangulating intestinal infarction associated with Strongylus vulgaris in referred Danish equine cases. Strongylus vulgaris is a pathogenic helminth parasite infecting horses and was once considered to be the primary cause of colic. Migrating larvae cause ischaemia and infarction of intestinal segments. This knowledge is derived from case reports and experimental inoculations of parasite-naïve foals, and it remains unknown to what extent the parasite is associated with different types of colic. Objective: To evaluate the role of S. vulgaris as a risk factor for different types of colic in horses. Methods: A retrospective case-control study among horses referred with abdominal pain to the Univ...
Impact of karyotype organization on interlocus recombination between T cell receptor genes in Equidae. The T cell receptor (TCR) genes (TRA, TRB, TRD and TRG) reside in 3 different chromosomal regions. During the maturation of T lymphocytes, the TCR genes are rearranged by site-specific recombination, a process that also predisposes T cells to aberrant rearrangements. Illegitimate recombination between the TCR genes occurs at a low level in healthy individuals, but this frequency may correlate with the risk of lymphoma. The aim of this work was to investigate interlocus recombination in equids. Illegitimate rearrangements were studied in peripheral blood lymphocytes by FISH with painting and BA...
The efficacy of ELISA commercial kits for the screening of equine infectious anemia virus infection. The most used and reliable indicator of Equine infectious anemia virus (EIAV) infection is the detection of its specific antibodies in horse serum. In the present study, the performance of two commercial ELISA tests for the detection of EIAV antibodies as well as the potential advantages of their use as an EIAV infection screening tool were evaluated in 302 horse serum samples. Both ELISA assays showed 100% diagnostic sensitivity, and 92.3-94.3% diagnostic specificity. Discordant results were analyzed by immunoblot. The results showed that both ELISA tests are very efficient at detecting EIAV ...
Hematologic and biochemical reference intervals for adult Friesian horses from North America. Established breed-specific reference intervals (RI) are an important tool for monitoring the health of horses. There is a lack of published work on breed-specific RI for Friesian horses. Objective: The goal of this project was to determine hematologic and biochemical RI for Friesian horses residing in North America. Methods: Strict inclusion and exclusion criteria were established for selection of reference subjects and for blood specimen collection and handling. Blood samples from 123 healthy, adult (range 3-18 years, median 8 years) Friesian horses of both sexes (70 mares, 45 geldings, and...
Development of a Microsphere-based Immunoassay for Serological Detection of African Horse Sickness Virus and Comparison with Other Diagnostic Techniques. African horse sickness (AHS) is a viral disease that causes high morbidity and mortality rates in susceptible Equidae and therefore significant economic losses. More rapid, sensitive and specific assays are required by diagnostic laboratories to support effective surveillance programmes. A novel microsphere-based immunoassay (Luminex assay) in which beads are coated with recombinant AHS virus (AHSV) structural protein 7 (VP7) has been developed for serological detection of antibodies against VP7 of any AHSV serotype. The performance of this assay was compared with that of a commercial enzyme-l...
Equine sperm-neutrophil binding. When mares are inseminated repeatedly, protein molecules from the seminal plasma (SP) prevent sperm-neutrophil binding and reduced fertility. The molecule(s) responsible for sperm-neutrophil binding is not known and the identification of beneficial SP proteins is complicated by their large numbers and abundant variation. We examined several important aspects of sperm-neutrophil binding to ultimately facilitate the identification and isolation of the molecule(s) responsible. First, we raised anti-equine P-selectin antibodies to determine the involvement of this adhesion molecule in sperm-neutro...
West Nile Virus Infection in Horses: Detection by Immunohistochemistry, In Situ Hybridization, and ELISA. This study describes the clinicopathologic findings in naturally occurring West Nile virus (WNV) infection in horses. WNV was diagnosed in a foal by immunohistochemical and in situ hybridization methods, and the presence of WNV antibodies was detected in 5 other horses with clinical signs suggestive of WNV infection. At necropsy of the foal, lymph nodes were edematous and enlarged, and the intestines showed diffuse congestion and focal hemorrhages. The most significant histologic lesions in this case were nonsuppurative meningoencephalomyelitis, particularly in the brainstem and spinal cord. I...
Novel monoclonal antibody against alphaX subunit from horse CD11c/CD18 integrin. The αX I-domain of the horse integrin CD11c was successfully expressed in Escherichia coli, purified, biochemically characterized and used as immunogen to generate murine monoclonal antibodies against horse CD11c, which are not yet commercially available. One monoclonal antibody mAb-1C4 against the αX I-domain, is an IgG2a able to interact with the recombinant I-domain, showing an EC50=2.4ng according to ELISA assays. By western blot with horse PBMCs lysates the mAb-1C4 recognized a protein of 150kDa which corresponds well with the CD11c molecule. Using immunohistochemistry in horse lymph no...
Rapid detection of equine coronavirus by reverse transcription loop-mediated isothermal amplification. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid detection of equine coronavirus (ECoV). This assay was conducted at 60 °C for 40 min. Specificity of the RT-LAMP assay was confirmed using several equine intestinal and respiratory pathogens in addition to ECoV. The novel assay failed to cross-react with the other pathogens tested, suggesting it is highly specific for ECoV. Using artificially synthesized ECoV RNA, the 50% detection limit of the RT-LAMP assay was 10(1.8)copies/reaction. This is a 50-fold greater sensitivity than conventio...
Pre-analytical stability of adrenocorticotrophic hormone from healthy horses in whole blood, plasma and frozen plasma samples. The stability of equine adrenocorticotrophic hormone (ACTH) in blood samples is not fully known. The study objectives were to determine ACTH stability (1) in whole blood and plasma over 72 h at either 4 or 21 °C, and (2) in plasma frozen at either -20 or -80 °C over 30 days. Nine horses were sampled and ACTH concentration were measured after storage as whole blood or plasma, at 4, 21, -20 and -80 °C for up to 30 days. The ACTH concentration was significantly reduced at 24 h but remained stable when plasma was frozen at -20 and -80 °C for 30 days. Beyond 24 h, samples stored a...
Hendra virus survival does not explain spillover patterns and implicates relatively direct transmission routes from flying foxes to horses. Hendra virus (HeV) is lethal to humans and horses, and little is known about its epidemiology. Biosecurity restrictions impede advances, particularly on understanding pathways of transmission. Quantifying the environmental survival of HeV can be used for making decisions and to infer transmission pathways. We estimated HeV survival with a Weibull distribution and calculated parameters from data generated in laboratory experiments. HeV survival rates based on air temperatures 24 h after excretion ranged from 2 to 10 % in summer and from 12 to 33 % in winter. Simulated survival across the distri...
Circulating immune complexes and markers of systemic inflammation in RAO-affected horses. The aim of this study was to investigate the levels of circulating immune complexes (CICs) and concentration of haptoglobin, fibrinogen and C-reactive protein in the serum of horses with recurrent airway obstruction and healthy controls. The study was conducted on a group of 14 adult Polish Konik horses, kept in uniform environmental and living conditions. Horses were divided into two groups: 7 horses were not affected by any respiratory problem (control group) and 7 horses had a history of recurrent airway obstruction (RAO) (study group). A clinical and laboratory evaluation, endoscopic exami...
Necrotizing Enteritis and Hyperammonemic Encephalopathy Associated With Equine Coronavirus Infection in Equids. Equine coronavirus (ECoV) is a Betacoronavirus recently associated clinically and epidemiologically with emerging outbreaks of pyrogenic, enteric, and/or neurologic disease in horses in the United States, Japan, and Europe. We describe the pathologic, immunohistochemical, ultrastructural, and molecular findings in 2 horses and 1 donkey that succumbed to natural infection with ECoV. One horse and the donkey (case Nos. 1, 3) had severe diffuse necrotizing enteritis with marked villous attenuation, epithelial cell necrosis at the tips of the villi, neutrophilic and fibrinous extravasation into th...
Effects of cycle stage and sampling procedure on interpretation of endometrial cytology in mares. The aim of this study was to ascertain if (1) the stage of reproductive cycle influences cytological results, (2) cytology obtained from an endometrial biopsy is more accurate than cytology derived using the cytobrush, and (3) different methods used for evaluation of cytological samples produce similar diagnostic results. Material was collected from 46 mares in estrus, 48 in diestrus and from 33 mares in anestrus. Smears were evaluated using two criteria. In criterion I, a total of 300cells were counted and the percentage of polymorphonuclear cells was recorded. In criterion II, the number of ...
Evaluation of contact activation of citrated equine whole blood during storage and effects of contact activation on results of recalcification-initiated thromboelastometry. To evaluate the degree of activation of the contact pathway in citrated equine whole blood over holding times ≤ 30 minutes and assess effects of contact activation on recalcification-initiated thromboelastometry. Methods: 11 healthy adult mixed-breed horses. Methods: Blood was collected by atraumatic jugular venipuncture into prewarmed evacuated siliconized glass tubes containing citrate anticoagulant and held at 37°C for ≤ 30 minutes. Thromboelastometry was performed with an in vitro viscoelasticity (thromboelastometry) monitoring system. Factor XII and factor XI procoagulant activities ...
