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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.
Phenotypical assays and partial sequencing of the hsp60 gene for identification of Streptococcus equi. Strangles is an acute and contagious disease characterized by inflammation of the upper respiratory tract of horses. The etiological agent of strangles is the bacteria S. equi subsp. equi, which belongs to the Lancefield group C. Opportunistic agents from the same group are frequently isolated from horses with strangles and may induce mistaken diagnoses. Among the subspecies of S. equi, the phenotypic features are almost undistinguishable; however, the pathogenic potential is widely differentiated. The aim of this study was to characterize S. equi isolates obtained from clinical samples of str...
Combinatorial selection of a RNA thioaptamer that binds to Venezuelan equine encephalitis virus capsid protein. A phosphorothioate RNA aptamer (thioaptamer) targeting the capsid protein of Venezuelan equine encephalitis virus (VEEV) was isolated by in vitro combinatorial selection. The selected thioaptamer had a strong binding affinity (approximately 7nM) and high specificity for the target protein. For the binding to the protein, the overall tertiary structure of the thioaptamer is required. We introduce two theoretical methods to examine the effect of phosphorothioate modification on the enhancement of binding affinity and one experimental method to examine the nature of the multiple bands of thioapta...
A preliminary study of the short circuit current (Isc) responses of sweat gland cells from normal and anhidrotic horses to purinergic and adrenergic agonists. The causal factors of equine anhidrosis have not yet been elucidated but defective electrolyte transport mechanisms in the gland are likely to be involved. To investigate this possibility, experiments were performed on cultured equine sweat gland epithelia from five free-sweating UK horses (3 intact males, 2 mares, aged 2-4 years) and from three free-sweating Singapore horses (1 intact male, 2 mares, aged 3-5 years) and three anhidrotic (Singapore) horses (1 intact male, 1 gelding, 1 mare, aged 3-6 years). Cultured cells from each animal were grown on permeable supports and loaded into Ussing ...
Cytokine and chemokine gene expression of IL-1beta stimulated equine articular chondrocytes. To evaluate mRNA expression of several proinflammatory and anti-inflammatory cytokines and chemokines in equine unstimulated and interleukin-1beta (IL-1beta)-stimulated chondrocytes. Methods: In vitro experiment using equine chondrocyte cultures. Methods: Whole articular cartilage from metacarpophalangeal joints (n=5 horses; 10 fetlocks). Methods: Chondrocyte monolayer cultures were established from digested adult equine articular cartilage and stimulated with 5 ng/mL of recombinant human IL-1beta. RNA was extracted from the cells 24 hours after stimulation. IL-1beta, IL-4, IL-6, IL-8, tumor n...
Infections caused by pathogenic free-living amebas (Balamuthia mandrillaris and Acanthamoeba sp.) in horses. This article describes amebic infections in 4 horses: granulomatous amebic encephalitis caused by Balamuthia mandrillaris and Acanthamoeba culbertsoni and systemic infections caused by Acanthamoeba sp. The former infection occurred in 1 of 4 horses spontaneously without any underlying conditions; the latter amebic infection was perhaps "opportunistic" considering the visceral involvement by this protozoan in association with Aspergillus sp. and/or Escherichia coli and Pseudomonas sp. The clinicopathologic findings and demonstration of the amebic organisms using immunohistochemical techniques, ...
Effects of blood contamination of cerebrospinal fluid on results of indirect fluorescent antibody tests for detection of antibodies against Sarcocystis neurona and Neospora hughesi. The purpose of this study was to determine the effect of blood contamination of cerebrospinal fluid (CSF) on the results of indirect fluorescent antibody tests (IFATs) for Sarcocystis neurona and Neospora hughesi. The in vitro study used antibody-negative CSF collected from non-neurologic horses immediately after euthanasia and blood samples from 40 healthy horses that had a range of IFAT antibody titers against S. neurona and N. hughesi. Serial dilutions of whole blood were made in seronegative CSF to generate blood-contaminated CSF with red blood cell (RBC) concentrations ranging from 10 to ...
Major retinal autoantigens remain stably expressed during all stages of spontaneous uveitis. Equine recurrent uveitis (ERU) is a valuable model for autoimmune diseases, since it develops frequently and occurs spontaneously. We investigated the overall expression level of three major retinal autoantigens in normal retinas and various ERU stages. Analysis of retinal proteomes of both, healthy and diseased retinas revealed an almost unaffected expression of IRBP, S-antigen and cRALBP in ERU cases. Validation of these findings with western blots and immunohistochemistry confirmed constant to increased expression of these autoantigens, although loss of their physiological expression sites ...
A quantitative PCR assay for the detection and quantification of Babesia bovis and B. bigemina. The haemoparasites Babesia bovis and Babesia bigemina affect cattle over vast areas of the tropics and temperate parts of the world. Microscopic examination of blood smears allows the detection of clinical cases of babesiosis, but this procedure lacks sensitivity when parasitaemia levels are low. In addition, differentiating between similar haemoparasites can be very difficult. Molecular diagnostic procedures can, however, overcome these problems. This paper reports a quantitative PCR (qPCR) assay involving the use of SYBR Green. Based on the amplification of a small fragment of the cytochrome...
Sequencing of cDNA and proximal promoter of equine hexokinase II gene. In order to investigate the utilization of glucose in equine skeletal muscle, we determined the coding and proximal promoter sequences of the hexokinase type II (HKII) gene in thoroughbred horse, Grevy's zebra and Hartmann's mountain zebra. The deduced amino acid sequence of thoroughbred horse HKII showed 100, 100, 94.4, 92.7 and 92.6% identities with Grevy's zebra, Hartmann's mountain zebra, human, mouse and rat HKIIs, respectively. In equine HKIIs, specific amino acid substitutions, Ile 159 and Arg 610, were found in the potential binding site for glucose. In addition, the nucleotide sequenc...
Potential risk of equine herpes virus 1 (EHV-1) transmission by equine embryo transfer. The objective of this study was to determine whether the 10 wash cycles proposed by the International Embryo Transfer Society (IETS) for bovine embryos efficiently decontaminated equine embryos exposed to equine herpes virus 1 (EHV-1) in vitro. Donor mares and stallions were individually screened and shown to be negative for the virus by PCR detection of EHV-1 DNA in blood leukocytes, semen, and uterine lavages in which embryos were recovered. Twenty embryos were recovered and randomly assigned to one of two groups: 10 embryos were exposed for 24h to infectious EHV-1 at 10(6)TCID(50)/ml, and 1...
Efficacy of 2,6-dichlorophenol lure to control Dermacentor nitens. This study was carried out with the objective of evaluating the efficacy of a 2,6-dichlorophenol (2,6-DCP) lure to control Dermacentor nitens (Acari: Ixodidae). Slow-release formulations of the pheromone formulated with and without cypermethrin were prepared. Olfactometer bioassays were used to define the best dose of the pheromone and to evaluate the effect of cypermethrin with 2,6-DCP attractiveness. Sexually active males were released 15 cm from 2 cmx1 cm pieces of polypropylene treated with different odors: 2,6-DCP in a liposphere system (1.5, 30 and 300 microg--without cypermethrin and 30...
Potential involvement of EGF-like growth factors and phosphodiesterases in initiation of equine oocyte maturation. Human chorionic gonadotropin (hCG) was administered to mares in estrus with large, dominant ovarian follicles to initiate follicular and oocyte maturation. Follicular contents were collected at 0, 2, 4 and 6 h after hCG. Epiregulin, amphiregulin and phosphodiesterase (PDE) mRNA contents of granulosa cells (PDE 4D) were determined by reverse transcription and real-time PCR; PDE 3A mRNA content of single oocytes was determined similarly. Copy numbers of mRNA did not increase for PDE 3A or 4D over the time interval studied. Amounts of epiregulin and amphiregulin mRNA were correlated (r=0.98) when...
Expression and nephron segment-specific distribution of major renal aquaporins (AQP1-4) in Equus caballus, the domestic horse. Aquaporins (AQPs) play fundamental roles in water and osmolyte homeostasis by facilitating water and small solute movement across plasma membranes of epithelial, endothelial, and other tissues. AQP proteins are abundantly expressed in the mammalian kidney, where they have been shown to play essential roles in fluid balance and urine concentration. Thus far, the majority of studies on renal AQPs have been carried out in laboratory rodents and sheep; no data have been published on the expression of AQPs in kidneys of equines or other large mammals. The aim of this comparative study was to determ...
Modification of host erythrocyte membranes by trypsin and chymotrypsin treatments and effects on the in vitro growth of bovine and equine Babesia parasites. In the present study, we investigated the effects of protease pretreatments of host erythrocytes (RBC) on the in vitro growth of bovine Babesia parasites (Babesia bovis and B. bigemina) and equine Babesia parasites (B. equi and B. caballi). The selected proteases, trypsin and chymotrypsin, clearly modified several membrane proteins of both bovine and equine RBC, as demonstrated by SDS-PAGE analysis; however, the protease treatments also modified the sialic acid content exclusively in bovine RBC, as demonstrated by lectin blot analysis. An in vitro growth assay using the protease-treated RBC sh...
Determination of lactate concentrations in blood plasma and peritoneal fluid in horses with colic by an Accusport analyzer. Intestinal hypoperfusion can lead to increased lactate concentrations in plasma and peritoneal fluid of horses with colic. Objective: The purposes of this study were to (1) evaluate the reliability of the Accusport analyzer to assess peritoneal fluid lactate (PFL) concentrations in healthy horses and those with colic, (2) identify clinical features associated with abnormal blood plasma lactate (BPL) and PFL concentrations, and (3) evaluate the prognostic value of BPL and PFL. Methods: BPL and PFL were determined in 20 healthy horses and in 106 horses with colic. Results: The Accusport was reli...
Automated liquid chromatography-tandem mass spectrometry method for the analysis of firocoxib in urine and plasma from horse and dog. A rugged, sensitive and efficient liquid chromatography-tandem mass spectrometry method was developed and validated for the quantitative analysis of firocoxib in urine from 5 to 3000 ng/mL and in plasma from 1 to 3000 ng/mL. The method requires 200 microL of either plasma or urine and includes sample preparation in 96-well solid phase extraction (SPE) plates using a BIOMEK 2000 Laboratory Automated Workstation. Chromatographic separation of firocoxib from matrix interferences was achieved using isocratic reversed phase chromatography on a PHENOMENEX LUNA Phenyl-Hexyl column. The mobile phase w...
Real-time PCR for detection and differentiation of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus. Strangles is a contagious equine disease caused by Streptococcus equi subsp. equi. In this study, clinical strains of S. equi (n=24) and Streptococcus equi subsp. zooepidemicus (n=24) were genetically characterized by sequencing of the 16S rRNA and sodA genes in order to devise a real-time PCR system that can detect S. equi and S. zooepidemicus and distinguish between them. Sequencing demonstrated that all S. equi strains had the same 16S rRNA sequence, whereas S. zooepidemicus strains could be divided into subgroups. One of these (n=12 strains) had 16S rRNA sequences almost identical with the...
A direct enzyme immunoassay for the measurement of furosemide in horse plasma. A new enzyme immunoassay (EIA) for the measurement of furosemide in horse plasma is described. The lower limit of detection of this EIA method was 7.8 ng/ml. The intra-and inter-assay coefficients of variation ranged from 2.5% to 4.9% and 7.5% to 9.8%, respectively. Cross-reactivity with other compounds was not observed. There was a high correlation (r2=0.987) between the high-performance liquid chromatography and EIA results obtained for furosemide concentrations in horse plasma. These results indicate that the newly developed EIA method is useful for the quantitative analysis of furosemide i...
Identification and functional analysis of sequence variants in the long control region and the E2 open reading frame of bovine papillomavirus type 1 isolated from equine sarcoids. BPV-1 DNA is the predominant viral type detected in equine sarcoids and represents the only reported natural cross species infection of papillomaviruses. In this study, nucleotide variations in the LCR and the E2 regions of equine sarcoid-associated BPV-1 were characterised by sequence analysis. Variants particular to sarcoid BPV-1 were identified in both the LCR and E2 sequence. The functionality of the most common LCR variant was examined in equine and bovine cells. These studies showed that the activity of the variant LCR was higher in equine cells than bovine cells; the activity of the var...
Myosin heavy chain fibre type composition in foals: analyses at the mRNA and protein level. An optimal developed musculoskeletal system is vital for the performance of the horse. Previously, we showed that in the m. gluteus medius from adult untrained horses, identical mRNA and protein expression patterns were found in the majority of fibres. However, co-expression of IIa and IId/x myosin heavy chain (MyHC) was substantially more common at the protein than at the mRNA level, suggesting a transcriptionally controlled fine-tuning of these 2 genes. Objective: To analyse the MyHC transcripts and proteins (including the cardiac alpha isoform) in the same muscle during post natal developme...
Gene expression profiling in equine muscle tissues using mouse cDNA microarrays. Progress could be achieved by using microarrays to understand metabolic adaptations and disorders in equine muscle in response to exercise. Objective: To test the feasibility of using mouse cDNA microarrays to analyse gene expression profile in normal equine muscles. Methods: Muscular biopsies of dorsal gluteus medius and longissimus lumborum were done in 4 healthy Standardbreds. Total RNA was extracted from the muscle samples. The concentration and quality of RNA were measured before and after amplification. Gene expression profiles were measured using mouse cDNA microarrays including 15,264 ...
Is the distribution of selenium and zinc in the sublocations of spermatozoa regulated? In the sperm nuclei, of mammalian species selenium has been found only in the form of sperm nuclei glutathione peroxidase (snGPx) where it is most likely bound to the chromatin of spermatozoa. Over 80% of selenium in sperm is bound to the selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) in the midpiece of rat sperm. Zinc in sperm is mainly contained in the outer dense fiber (ODF) proteins of the flagella of mammalian spermatozoa. In the sperm nuclei, zinc is predominately located in the chromatin to the protamine proteins. In order to investigate if the insertion of zinc...
Hypothermic storage of equine isolated hepatocytes. The aim of the study was to establish the optimal methods for hypothermic storage of equine isolated hepatocytes. Viability of equine isolated hepatocytes after hypothermic storage was dependent on the type of storage medium as well as on the cell density in the storage suspension and the preservation period. Hepatocytes stored at 4 degrees C in Hanks' Balanced Salt Solution (HBSS) and Williams' Medium E (WE) for 24 h showed very low viability, numerous cell membrane blebs, very low attachment rate (11.9 +/- 6.5% and 34.8 +/- 19.1%, respectively) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte...
Biogas production with horse dung in solid-phase digestion systems. Experiments on methanogenesis from horse dung were conducted in laboratory-scale batch reactors in order to determine the substrate performance in a solid-phase digestion process, more specifically in terms of potential energy recovery and suitable process technology. Dung from a horse stable with straw bedding was used. The temperature was kept in the mesophilic range. In the percolation process (with process water sprinkled over the stacked biomass) a proportion of 10-20% of solid inoculum (pre-digested horse dung) was found to be suitable. Comparative experiments with both percolation and f...
Mesenchymal stem cell therapy in equine musculoskeletal disease: scientific fact or clinical fiction? The goal in the therapeutic use of mesenchymal stem cells (MSCs) in musculoskeletal disease is to harness the regenerative nature of these cells focussing on their potential to grow new tissues and organs to replace damaged or diseased tissue. Laboratory isolation of MSCs is now well established and has recently been demonstrated for equine MSCs. Stem cell science has attracted considerable interest in both the scientific and clinical communities because of its potential to regenerate tissues. Research into the use of MSCs in tissue regeneration in general reflects human medical needs, however...
Development of a real-time RT-PCR assay for improved detection of Borna disease virus. Borna disease virus (BDV) is a non-segmented, negative-stranded RNA virus, which infects cells of the central nervous system (CNS) in many different species. BDV is the causative agent of the neurological disorders in horses and sheep termed classical Borna disease (BD), as well as staggering disease in cats. At present, the diagnosis staggering disease or feline BD is made by histopathology or immunohistochemistry of the CNS. In order to obtain a better clinical diagnostic tool, a duplex real-time RT-PCR assay (rRT-PCR) was developed. TaqMan probes and primers specific for the BDV P and BDV L...
Pharmacokinetics of boldenone and stanozolol and the results of quantification of anabolic and androgenic steroids in race horses and nonrace horses. Anabolic steroids (ABS) boldenone (BL; 1.1 mg/kg) and stanozolol (ST; 0.55 mg/kg) were administered i.m. to horses and the plasma samples collected up to 64 days. Anabolic steroids and androgenic steroids (ANS) in plasma were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The limit of detection of all analytes was 25 pg/mL. The median absorption (t1/2 partial differential) and elimination (t1/2e) half-lives for BL were 8.5 h and 123.0 h, respectively, and the area under the plasma concentration-time curve (AUCho) was 274.8 ng.h/mL. The median t1/2e for ST was 82.1 ...
