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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.
Characterising Non-Structural Protein NS4 of African Horse Sickness Virus. African horse sickness is a serious equid disease caused by the orbivirus African horse sickness virus (AHSV). The virus has ten double-stranded RNA genome segments encoding seven structural and three non-structural proteins. Recently, an additional protein was predicted to be encoded by genome segment 9 (Seg-9), which also encodes VP6, of most orbiviruses. This has since been confirmed in bluetongue virus and Great Island virus, and the non-structural protein was named NS4. In this study, in silico analysis of AHSV Seg-9 sequences revealed the existence of two main types of AHSV NS4, designat...
Expression of oxytocin receptors is greatly reduced in the placenta of heavy mares with retained fetal membranes due to secondary uterine atony. Fetal membrane retention can be a life-threatening condition and its incidence exceeds 50% in heavy draught mares. Although fetal membrane retention is commonly treated with repeated injections of oxytocin, based on the suggestion that it is caused mainly by secondary atony of the uterus, this treatment sometimes fails. This led us to ask if expression of oxytocin receptors differs in mares that retain fetal membranes due to secondary uterine atony. Objective: To determine whether expression of oxytocin receptors in equine placental tissues differs when heavy draught mares expel fetal membrane...
Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites. A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equ...
Efficacy of Mechanical versus Non-Mechanical Sterile Preoperative Skin Preparation With Chlorhexidine Gluconate 4% Solution. To compare the efficacy of sterile preoperative skin antisepsis using either a 5-minute mechanical preparation or 5-minute non-mechanical preparation with chlorhexidine gluconate 4% solution. Methods: Matched design, ANOVA. Methods: Healthy adult Thoroughbred horses (n = 30). Methods: Each horse had both surgical preparation methods randomly assigned to identical sites on the left or right upper thigh. Prepared sites were sampled and cultured for bacteria after each preparation step. Results: Mechanical and non-mechanical preparation techniques significantly reduced bacteria isolated from ...
Rapid and accurate identification of Streptococcus equi subspecies by MALDI-TOF MS. Streptococcus equi includes very important animal and human pathogens. S. equi subsp. equi (SEE) is a highly pathogenic equine specific subspecies, while S. equi subsp. zooepidemicus (SEZ) and S. equi subsp. ruminatorum are opportunistic pathogens of various animal species and humans. Due to great phenotypic and sequence similarity between three subspecies their discrimination remains difficult. In this study, we aimed to design and validate a novel, Superspectra based, MALDI-TOF MS approach for reliable, rapid and cost-effective identification of SEE and SEZ, the most frequent S. equi subspec...
An inflammatory equine model demonstrates dynamic changes of immune response and cartilage matrix molecule degradation in vitro. The molecular aspects of inflammation were investigated in equine articular cartilage explants using quantitative proteomics. Articular cartilage explants were stimulated with interleukin (IL)-1β in vitro for 25 days, and proteins released into cell culture media were chemically labeled with isobaric mass tags and analyzed by liquid chromatography-tandem mass spectrometry. A total of 127 proteins were identified and quantified in media from explants. IL-1β-stimulation resulted in an abundance of proteins related to inflammation, including matrix metalloproteinases, acute phase proteins, comp...
Cholestanol-loaded-cyclodextrin improves the quality of stallion spermatozoa after cryopreservation. This study was to compare the effect of adding cholesterol or cholestanol loaded cyclodextrins in stallion sperm prior to cryopreservation to optimize sperm cryosurvival. Ejaculates from each of eight stallions were diluted to 120 million cells in a S-MEDIUM diluent. The diluted sperm were sub-divided into three treatments: no additive (control); 0.75mg of cyclodextrin pre-loaded with cholesterol (CLC)/120 million sperm (positive control); 1.5mg CLC/120 million sperm; 0.75mg of cyclodextrin pre-loaded with cholestanol (CnLC)/120 million sperm; and 1.5mg CnLC/120 million sperm. To set the exper...
Identification of phlebotomine sand fly blood meals by real-time PCR. Phlebotomine sand flies are blood-feeding insects of great medical and veterinary significance acting as vectors of Leishmania parasites. Studying the blood-feeding pattern of these insects may help in the understanding of their interactions with potential reservoir hosts of Leishmania parasites. In this study, we developed real time PCR assays for the identification of sand fly blood meal. Methods: Six pairs of primers were designed based on cytochrome b gene sequences available in GenBank of the following potential hosts: dog, cat, horse, chicken, black rat, and human. Firstly, SYBR Green-ba...
Serological evidence of equine influenza infections among persons with horse exposure, Iowa. Equine influenza virus (EIV) is considered enzootic in North America and experimental studies have documented human EIV infections. Methods: This cross-sectional study examined 94 horse-exposed and 34 non-exposed controls for serological evidence of EIV infection. Sera were evaluated for antibodies against three EIV and two human H3N2 viruses using microneutralization (MN), neuraminidase inhibition (NI), enzyme-linked lectin (ELLA), and hemagglutination inhibition (HI) serological assays. Risk factor analyses were conducted using logistic regression and proportional odds modeling. Results: The...
Transgenically expressed Parascaris P-glycoprotein-11 can modulate ivermectin susceptibility in Caenorhabditis elegans. P-glycoproteins (Pgps) are suspected to mediate drug extrusion in nematodes contributing to macrocyclic lactone resistance. This association was recently shown for Parascaris Pgp-11. Ivermectin resistance was correlated with the presence of three pgp-11 single nucleotide polymorphisms and/or increased pgp-11 mRNA levels. In the present study, the ability of Pgp-11 to modulate ivermectin susceptibility was investigated by its expression in a pgp-11-deficient Caenorhabditis elegans strain. Expression of Parascaris pgp-11 in two transgenic lines significantly decreased ivermectin susceptibility i...
[Flow-cytofluorometric study of bactericidal granules in blood phagocytes of animals with various species sensitivity to experimental plague infection]. Compare the content of bactericidal granules (BG) in blood phagocytes of animals, that differ by species sensitivity to plague infection, under the conditions of measuring, that ensure automatic differentiating by this parameter of monocytes and granulocytes of human blood. Methods: Human whole blood leukocytes were studied, as well as from 7 animal species: mice, guinea pigs, golden hamsters, white rats, rabbits, dogs and horses. Acridine orange (AO) was used for supra-vital staining in primary (bactericidal) granule cells. Relative BG content was measured in separate cells in conventional un...
Pharmacokinetics of intra-articular betamethasone sodium phosphate and betamethasone acetate and endogenous hydrocortisone suppression in exercising horses. To the date, no reports exist of the pharmacokinetics (PK) of betamethasone (BTM) sodium phosphate and betamethasone acetate administered intra-articular (IA) into multiple joints in exercising horses. The purpose of the study was to determine the PK of BTM and HYD concentrations in plasma and urine after IA administration of a total of 30 mg BTM. Eight 4 years old Thoroughbred mares were exercised on a treadmill and BTM was administered IA. Plasma and urine BTM and HYD were determined via high performance liquid chromatography spectrometry for 6 weeks. Concentration-time profiles of BTM and H...
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 ...
