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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.
Study on membrane fluidity and erythrocyte aggregation in equine, bovine and human species. The aim of the present paper is to analyze whether membrane fluidity can be predicted from its lipid composition and to assay the possible relationship between such variable and the aggregating properties of erythrocytes from equine, bovine and human species due to the widely acknowledged differences in their tendency to form aggregates. The main difference between phospholipids from plasma membrane in these species lies in the concentration levels of sphyngomyelin (SM) and phosphatidilcoline (PC); more precisely, in the external hemilayer of the lipid bilayer. Membrane fluidity was estimated ...
A chromosome inversion near the KIT gene and the Tobiano spotting pattern in horses. Tobiano is a white spotting pattern in horses caused by a dominant gene, Tobiano(TO). Here, we report TO associated with a large paracentric chromosome inversion on horse chromosome 3. DNA sequences flanking the inversion were identified and a PCR test was developed to detect the inversion. The inversion was only found in horses with the tobiano pattern, including horses with diverse genetic backgrounds, which indicated a common genetic origin thousands of years ago. The inversion does not interrupt any annotated genes, but begins approximately 100 kb downstream of the KIT gene. This inversion...
A defined medium supports changes consistent with capacitation in stallion sperm, as evidenced by increases in protein tyrosine phosphorylation and high rates of acrosomal exocytosis. Efficient in vitro capacitation of stallion sperm has not yet been achieved, as suggested by low sperm penetration rates reported in in vitro fertilization (IVF) studies. Our objectives were to evaluate defined incubation conditions that would support changes consistent with capacitation in stallion sperm. Protein tyrosine phosphorylation events and the ability of sperm to undergo acrosomal exocytosis under various incubation conditions were used as end points for capacitation. Sperm incubated 4-6h in modified Whitten's (MW) with the addition of 25 mM NaHCO3 and 7 mg/mL BSA (capacitating mediu...
Endometrial explant culture for characterizing equine endometritis. Endometritis after insemination is ubiquitous in the horse and is associated with semen and/or bacteria in the uterus. In up to 40% of horses, inflammation persists causing infertility. An endometrial explant culture was developed to study uterine secretion of prostaglandin F(2alpha) (PGF(2alpha)) in response to physiological and pathological challenge. Methods: Uteri were collected from mares, the endometrium dissected and explants from the uterine body or horn cultured in William's or RPMI medium. The response of explants to oxytocin, semen or bacteria compared to untreated tissue was tested...
Evaluation of equine laminar vein function: harvesting, dissection and the use of functional methods to distinguish between veins and arteries. Pharmacological evaluation of the unique equine laminar microvasculature is crucial to understanding its role in health and in diseases such as laminitis. However, separating the distinctive characteristics of arterial versus venous components of this complex vascular network has previously proved to be extremely difficult. Encased in a hard hoof capsule, isolation of individual blood vessels presents a considerable challenge. Exacerbating this difficulty, the laminar venous network is adapted to sustain high intravascular pressures and consequently has thickened walls, making the normally str...
Circulating fatty acid profiles in response to three levels of dietary omega-3 fatty acid supplementation in horses. Fatty acids of the n-3 type confer health benefits to humans and other species. Their importance to equine physiology could include improved exercise tolerance, decreased inflammation, and improved reproductive function. The circulating fatty acid profile and the acquisition and washout of fatty acids in response to n-3 supplementation were determined for horses in the current study. A fatty acid supplement high in eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid was fed to deliver EPA plus DHA at 0 (control), 10, 20, or 40 g/d to 16 mares (n = 4/group) for 28 d. Plasma was collected at -...
Insulins in equine urine: qualitative analysis by immunoaffinity purification and liquid chromatography/tandem mass spectrometry for doping control purposes in horse-racing. Insulin is a peptide hormone consisting of two peptide chains (A- and B-chain) that are cross-linked by two disulfide bonds. To obtain improved pharmacokinetic onset of action profiles of insulin treatment in diabetic patients, recombinant long-, intermediate-, and rapid-acting insulin analogs are produced, in which the C-terminal end of the B-chain plays an especially important role.A review of the veterinary literature reveals the low prevalence of equine type I diabetes mellitus, which indicates that the therapeutic use of insulin in racing horses is unlikely. Although there is no unequivoc...
A comparison of coprological, serological and molecular methods for the diagnosis of horse infection with Anoplocephala perfoliata (Cestoda, Cyclophyllidea). Anoplocephala perfoliata (Cestoda, Cyclophyllidea), the commonest intestinal tapeworm of horses, can cause colic, intussusceptions, ileal impactions and intestinal perforations. Common diagnostic techniques for A. perfoliata infection, i.e. coprology and serology, show inherent limitations in terms of sensitivity and specificity and new approaches are thus required. Hence, the present study compared the reliability of coprological, serological (i.e. ELISA) and molecular (i.e. nested PCR) methods in detecting A. perfoliata infection in naturally infected horses and in horses treated with a comb...
Streptococcus henryi sp. nov. and Streptococcus caballi sp. nov., isolated from the hindgut of horses with oligofructose-induced laminitis. 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...
In vitro efficacy of an ophthalmic drug combination against corneal pathogens of horses. To evaluate the in vitro efficacy of an ophthalmic drug combination against common corneal pathogens of horses. Methods: Representative isolates of 3 bacterial and 2 fungal corneal pathogens of horses. Methods: Pathogens were subjected to minimum inhibitory concentration (MIC) testing of a drug combination that consisted of equal volumes of natamycin 3.33%, tobramycin 0.3%, cefazolin 5.5%, and equine serum. Proteinase inhibitory activity of the drug combination was assessed by use of a fluorescence microplate assay with gelatin and collagen I as substrates. The MICs of the drug combination wer...
In vitro cell compatibility and antibacterial activity of microencapsulated doxycycline designed for improved localized therapy of septic arthritis. For the treatment of septic arthritis in large animals, the local application of antibiotics as a slow release system may be an appropriate means to reach high local bioactivity and low systemic side effects and drug residues. In this study, doxycycline microspheres were developed and tested in vitro for their drug-release properties, suitability for intra-articular application and antimicrobial activity. Methods: The development of a slow release system was achieved by microencapsulation of the drug into poly(lactide-co-glycolide) microspheres by a novel ultrasonic atomization method. Drug el...
Platelet activation in ponies with airway inflammation. Platelet activation occurs in human obstructive airway diseases and in laboratory animal models. However, there is limited evidence that platelets may be involved in equine recurrent airway obstruction (RAO) and other inflammatory diseases. This study investigated whether platelet activation also occurred in RAO. Objective: Platelet function is altered in ponies with active RAO. This alteration can be detected ex vivo by measuring platelet adhesion. Methods: An in vitro platelet adhesion assay measuring acid phosphatase (AcP) activity colorimetrically was adapted for use with equine platelets ...
Interpretation of serum antibody response to Anoplocephala perfoliata in relation to parasite burden and faecal egg count. Increased knowledge is needed to assist in the interpretation of presently available diagnostic techniques for infection by the tapeworm Anoplocephala perfoliata in horses. Objective: The suggested cut-off level of an A. perfoliata specific ELISA may not adequately reflect the actual infection level. Hence, faecal egg counts may be a more useful diagnostic test for individual horses than previously reported. Methods: Eighty-four horses admitted for slaughter at a Danish abattoir were examined for the presence of A. perfoliata. The number of tapeworms, their stage of development and gross patho...
Phosphodiesterase isoenzymes in equine platelets and their influence on platelet adhesion. To determine the phosphodiesterase (PDE) isoenzymes in equine platelets and evaluate their influence on platelet adhesion. Methods: Platelets obtained from healthy New Forest Pony geldings that ranged from 12 to 20 years of age (mean +/- SEM, 17.3 +/- 1.1 years). Methods: PDE isoenzyme activity in equine platelets was determined by use of a 2-step radioactive assay. Functional importance of PDE isoenzymes was established by use of selective inhibitors in a colorimetric adhesion assay. Results: PDE1, PDE2, PDE3, and PDE5 and small amounts of PDE4 were found in equine platelets. Inhibition of PD...
Construction of a recombinant plasmid as reaction control in routine PCR for detection of contagious equine metritis (CEM-PCR). Contagious equine metritis (CEM) is a highly contagious bacterial venereal disease of horses caused by Taylorella equigenitalis. CEM-PCR is a semi-nested PCR method for detecting this bacterium. Although this technique is regarded as a sensitive diagnostic method for CEM, there are risks of it generating false positive and false negative results. In this study, we constructed a recombinant plasmid (CEM-POS) as reaction control to assure adequate PCR reaction and prevent false positive results caused by contamination of the reaction control in routine CEM-PCR examinations. CEM-POS was construct...
Loss of expression of protein kinase a regulatory subunit 1alpha in pigmented epithelioid melanocytoma but not in melanoma or other melanocytic lesions. Pigmented epithelioid melanocytoma (PEM) is a recently described entity comprising most cases previously described as "animal-type melanoma" and epithelioid blue nevus (EBN) occurring in patients with the multiple neoplasia syndrome Carney complex (CNC). Mutations of the protein kinase A regulatory subunit type 1alpha (R1alpha) (coded by the PRKAR1A gene) are found in more than half of CNC patients. In this study, we investigated whether PEM and EBN are related at the molecular level, and whether changes in the PRKAR1A gene status and the expression of the R1alpha protein may be involved in th...
Equine coronavirus induces apoptosis in cultured cells. Equine coronavirus (ECoV) was first isolated from a diarrheic foal and was found genetically similar to group II coronaviruses. However, its pathological characteristics were not adequately investigated. In our preliminary in vitro investigation, ECoV-induced cell death was observed in bovine kidney-derived MDBK cells. Based on this finding, we investigated whether the ECoV-induced CPE was apoptosis. Following ECoV infection, MDBK cells showed morphological changes such as cell rounding and detachment from the culture surface. Moreover, syncytium formation was observed as the other type of cyt...
Equine infectious anemia virus entry occurs through clathrin-mediated endocytosis. Entry of wild-type lentivirus equine infectious anemia virus (EIAV) into cells requires a low-pH step. This low-pH constraint implicates endocytosis in EIAV entry. To identify the endocytic pathway involved in EIAV entry, we examined the entry requirements for EIAV into two different cells: equine dermal (ED) cells and primary equine endothelial cells. We investigated the entry mechanism of several strains of EIAV and found that both macrophage-tropic and tissue culture-adapted strains utilize clathrin-coated pits for entry. In contrast, a superinfecting strain of EIAV, EIAV(vMA-1c), utilizes ...
Cloning, expression and purification of envelope proteins E1 and E2 of western equine encephalitis virus and potential use of them as antigens in immunoassays. The genes encoding envelope proteins E1 and E2 of western equine encephalitis virus (WEEV) were respectively cloned into a prokaryotic T7 RNA polymerase-regulated expression vector. The recombinant C-terminal 6xHis-tagged WEEV E1 and E2 were expressed in bacteria as inclusion bodies that were subsequently solubilized with 8M urea, purified by immobilized metal ion affinity chromatography and finally refolded using an arginine system. The purified 6xHis-tagged proteins showed 50kDa bands as revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, consistent with the expected size...
Lactobacillus hayakitensis sp. nov., isolated from intestines of healthy thoroughbreds. Two strains, KBL13(T) and GBL13, were isolated as one of intestinal lactobacilli from the faecal specimens from different thoroughbreds of the same farm where they were born in Hokkaido, Japan. They were Gram-positive, facultatively anaerobic, catalase-negative, non-spore-forming and non-motile rods. KBL13(T) and GBL13 homofermentatively metabolize glucose, and produce lactate as the sole final product from glucose. The 16S rRNA gene sequence, DNA-DNA hybridization, DNA G+C content and biochemical characterization indicated that these two strains, KBL13(T) and GBL13, belong to the same species...
Analysis of ORFs 2b, 3, 4, and partial ORF5 of sequential isolates of equine arteritis virus shows genetic variation following experimental infection of horses. Samples from horses experimentally infected with the "large plaque variant (LP3A+)" of equine arteritis virus were analysed. These included 182 nasal swabs collected from day 1 to 14 post-infection (p.i.), and 21 virus isolates obtained from white blood cells of animals that showed a prolonged viraemia between days 30 to 72 p.i. In order to determine the genetic stability of the virus and particularly to characterise the genetic variants found during the prolonged viraemia, partial sequences of open reading frame 5 (ORF5) encoding glycoprotein 5 (GP5) were generated. Viruses with amino acid su...
Isolation of bicarbonate from equine urine for isotope ratio mass spectrometry. Sodium bicarbonate administration to horses prior to competition in order to enhance the buffer capacity of the organism is considered as a doping offence. The analysis of the isotopic composition of urinary bicarbonate/CO(2) (TCO(2)) may help to identify an exogenous bicarbonate source, as technical sodium bicarbonate exhibits elevated delta(13)C values compared with urinary total carbon. The isolation of TCO(2) from 60 equine urine samples as BaCO(3) followed by an isotopic analysis shows a significant variability of delta(13)C for TCO(2) of more than 10 per thousand. The delta(13)C of total...
Comparison of five real-time PCR assays for detecting virulence genes in isolates of Escherichia coli from septicaemic neonatal foals. Fifty-five isolates of Escherichia coli from septicaemic neonatal foals were used to validate five real-time pcr assays targeting different known virulence factor genes: curli fibre (csgD), ferric hydroxamate uptake (fhuA), type 1A pilin (fimA), aerobactin (lutA) and yersiniabactin (fyuA). A pcr assay targeting a universal sequence of the bacterial 16S rrna gene served as quality control. The pcr assays showed good analytical specificity and sensitivity on the basis of sequencing the pcr products, their lack of cross-reactivity with non-E coli organisms, high amplification efficiency and a lim...
Expression of progesterone and oestrogen receptors by early intrauterine equine conceptuses. Progesterone and oestrogen play essential roles in the maintenance of pregnancy in eutherian mammals and are thought to exert their effects on the developing conceptus indirectly, via the endometrium. In some species, early embryos have themselves been shown to express steroid receptors, thereby suggesting that reproductive steroids may also influence embryonic development directly. The aim of this study was to determine whether early intrauterine equine conceptuses express either the classical intracellular progesterone (PR) and oestrogen receptors (ERalpha and ERbeta) or the more recently ch...
Mapping of equine lentivirus receptor 1 residues critical for equine infectious anemia virus envelope binding. The equine lentivirus receptor 1 (ELR1), a member of the tumor necrosis factor receptor (TNFR) protein family, has been identified as a functional receptor for equine infectious anemia virus (EIAV). Toward defining the functional interactions between the EIAV SU protein (gp90) and its ELR1 receptor, we mapped the gp90 binding domain of ELR1 by a combination of binding and functional assays using the EIAV SU gp90 protein and various chimeric receptor proteins derived from exchanges between the functional ELR1 and the nonbinding homolog, mouse herpesvirus entry mediator (murine HveA). Complement...
Development of a fluorescent-microsphere immunoassay for detection of antibodies specific to equine arteritis virus and comparison with the virus neutralization test. The development and validation of a microsphere immunoassay (MIA) to detect equine antibodies to the major structural proteins of equine arteritis virus (EAV) are described. The assay development process was based on the cloning and expression of genes for full-length individual major structural proteins (GP5 amino acids 1 to 255 [GP5(1-255)], M(1-162), and N(1-110)), as well as partial sequences of these structural proteins (GP5(1-116), GP5(75-112), GP5(55-98), M(88-162), and N(1-69)) that constituted putative antigenic regions. Purified recombinant viral proteins expressed in Escherichia col...
Diagnostic real-time PCR assay for the quantitative detection of Theileria equi from equine blood samples. We developed a TaqMan real-time polymerase chain reaction (PCR) assay for the quantitative detection of Theileria equi from the in vitro-cultured parasite and field blood samples collected from horses living in Ghana and Brazil. The detection limit for the assay was determined to be 1.5 parasites/microl per sample, and the quantitative capacity was demonstrated using the in vitro-cultured parasite. For field applications, the real-time PCR assay was compared to a previously established nested PCR assay used as the gold standard for the real-time PCR assay. Of 65 field blood samples, 46 samples...
Comprehensive screening of acidic and neutral drugs in equine plasma by liquid chromatography-tandem mass spectrometry. A multi-target high-throughput liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the detection of low ppt to low ppb levels of anabolic steroids, corticosteroids, anti-diabetics, and non-steroidal anti-inflammatory drugs (NSAIDs) in equine plasma was developed for the purpose of doping control. Plasma samples were first deproteinated by addition of trichloroacetic acid. Drugs were then extracted by solid-phase extraction (SPE) using Bond Elut Certify cartridges, and the extracts were analysed by a triple-quadrupole/linear ion trap LC-MS-MS instrument in positive electrospray...
Detection of Leptospira interrogans DNA and antigen in fixed equine eyes affected with end-stage equine recurrent uveitis. Equine recurrent uveitis (ERU) is the most frequent cause of blindness in horses worldwide. Leptospira has been implicated as an etiologic agent in some cases of ERU and has been detected in fresh ocular tissues of affected horses. The objective of this study was to determine the presence of Leptospira antigen and DNA in fixed equine ocular tissues affected with end-stage ERU. Sections of eyes from 30 horses were obtained. Controls included 1) 10 normal equine eyes and 2) 10 equine eyes with a nonrecurrent form of uveitis. The experimental group consisted of 10 eyes diagnosed with ERU based on...
Histomonas meleagridis (Parabasala, Trichomonadea, Monocercomonadidae): presence of natural agglutinins in horse serum. Cultured Histomonas meleagridis cells were readily agglutinated in vitro by horse serum at concentrations as low as 5%, although clumping was more rapid and prominent at 15% or higher. For observation of clumping, the cultured organisms were washed twice in Hanks balanced solution (HBSS) by centrifugation (1,000 x g for 15 min) and filtered through glass wool. The test sera were added and the mixture incubated in a Petri plate or 24-well culture plates at r.t. for 15-30 min. Formation of clumps was time- and concentration-dependent. Gentle agitation hindered agglutination at low serum concentr...
