Omneity® Pellets
All-In-One Vitamin & Mineral Pellet
Topic:Real-Time PCR
Real-Time PCR (Polymerase Chain Reaction) is a molecular technique used to amplify and quantify DNA sequences in horses. This method allows for the detection and measurement of specific genetic material in real-time, providing valuable insights into genetic expression, pathogen presence, and disease diagnosis. In equine research, Real-Time PCR is utilized to study various aspects such as infectious diseases, genetic disorders, and gene expression profiles. The technique's sensitivity and specificity enable researchers to accurately assess the genetic material of interest, facilitating advancements in equine health diagnostics and management. This page compiles peer-reviewed research studies and scholarly articles that explore the applications, methodologies, and findings related to Real-Time PCR in equine science.
RT-qPCR comparison of mast cell populations in whole blood from healthy horses and those with laminitis. Inflammatory damage to the digital laminae, a structure responsible for suspension of the distal skeleton within the hoof capsule, results in a painful and often life-threatening disease in horses called laminitis. There can be many diverse causes of laminitis; however, previous work in the horse has suggested that in each case, the inflammation and resulting tissue damage is consistent with the action of mediators released from mast cells (MC), as well as the downstream consequences of their activation. The recent development of molecular genetics tools to characterize cells based on their tr...
Microarray analysis after strenuous exercise in peripheral blood mononuclear cells of endurance horses. It is known that moderate physical activity may have beneficial effects on health, whereas strenuous effort induces a state resembling inflammation. The molecular mechanisms underlying the cellular response to exercise remain unclear, although it is clear that the immune system plays a key role. It has been hypothesized that the physio-pathological condition that develops in athletes subjected to heavy training is caused by derangement of cellular immune regulation. The purpose of the present study was to obtain information on endurance horse gene transcription under strenuous conditions and t...
Cloning and tissue expression of the equine transferrin receptor. Characterization of anemia in horses presents a challenge, as they do not release reticulocytes into peripheral blood. Transferrin receptor (TfR) expression is highest on erythroid cells in people and rats, and measurement of a soluble serum form (sTfR) is used to quantify erythropoiesis in these species. We hypothesized that equine TfR (eTfR) expression is similar in quantity and distribution to that in these other species and thus has potential for characterization of the regenerative response in anemic horses. Objective: This study was conducted to clone and sequence the eTfR gene and measu...
Real-time PCR and typing of Clostridium difficile isolates colonizing mare-foal pairs. Clostridium difficile infection can occur in the dams of sick foals, but it is unknown if mares and foals share the same isolates. In this study, C. difficile isolates from fecal samples of 11 mares paired with 11 foals were genotyped by arbitrarily primed PCR; two mares and three foals in five mare-foal pairs had diarrhea. Fecal immunoassays were utilized to detect C. difficile common antigen and toxin A. Quantitative real-time PCR (qPCR) systems were developed to detect genes for toxins A and B, as well as for binary toxin B. Sequences of all toxins were present in all isolates, although onl...
Temporal gene expression in equine corpora lutea based on serial biopsies in vivo. A biopsy procedure was developed to enable repeated sampling of a single equine corpus luteum (CL) over the course of an estrous cycle. The tissue collected was utilized in characterizing mRNA abundance for genes involved in luteal formation, function, and regression in the cyclic mare. Serial biopsies of CL in cyclic mares (2.7 to 27.5 mg per biopsy) were collected using an ultrasound-guided transvaginal technique. Biopsies were collected from each mare on d 2 and 5 (d 0 = ovulation) of the estrous cycle, and every other day from d 12 through luteolysis. Samples were obtained from 4 mares wit...
Description of the first recorded major occurrence of equine viral arteritis in France. The vast majority of equine arteritis virus (EAV) infections are inapparent or relatively mild, but may occasionally cause outbreaks of equine viral arteritis. The event observed in France during the summer of 2007 was the most important seen in the country, with mortality and disruption of economic activity. Objective: To describe the different stages seen during the outbreak and to show how molecular tools were used for both the detection and management of the crisis. Methods: EAV detection was performed by real-time reverse transcription-polymerase chain reaction (RT-PCR) in blood, nasal sw...
Regulation of SOX9 in normal and osteoarthritic equine articular chondrocytes by hyperosmotic loading. SOX9 is a transcription factor that is essential for cartilage extracellular matrix (ECM) formation. Osteoarthritis (OA) is characterised by a loss of cartilage ECM. In chondrocytes SOX9 gene expression is regulated by osmotic loading. Here we characterise SOX9 mRNA regulation through static and cyclical application of hyperosmotic conditions in normal and OA monolayer equine chondrocytes. Furthermore, we investigate whether extracellular signal-regulated protein kinase (ERK)1/2 mitogen-activated protein kinases (MAPK) pathways have a role in this regulation of SOX9. Methods: Equine chondrocyt...
Genetic diversity of piroplasms in plains zebra (Equus quagga burchellii) and Cape mountain zebra (Equus zebra zebra) in South Africa. Seventy EDTA blood samples collected from plains zebra (Equus quagga burchellii) and Cape mountain zebra (Equus zebra zebra) were screened for the presence of piroplasm parasite DNA using quantitative T. equi-specific and B. caballi-specific TaqMan real-time PCR (qPCR) tests. T. equi parasite DNA was detected in 60 samples, 19 of which were also positive for B. caballi. Approximately 1480bp of the piroplasm 18S rRNA gene was amplified and sequenced from 17 samples, while the V4 hypervariable region of the 18S rRNA gene was amplified, cloned and sequenced from 31 samples. BLASTN analysis reveal...
Is FAS/Fas ligand system involved in equine corpus luteum functional regression? Proapoptotic factor Fas ligand (FASL) and its cell surface receptor FAS are tumor necrosis factor superfamily members that trigger apoptosis in different cell types. However, their influence on luteal steroidogenesis is not clearly understood. The aim of the present work was to determine (i) the presence of the cytokine FASL and its receptor FAS in the mare's corpus luteum (CL) throughout the luteal phase, as well as (ii) the influence of FASL alone, or together with the cytokines tumor necrosis factor alpha (TNF) and interferon gamma (IFNG), on equine luteal cell production of luteotrophic an...
Evaluation of genes involved in prostaglandin action in equine endometrium during estrous cycle and early pregnancy. The aim was to evaluate expression of genes involved in the biosynthesis of prostaglandins (PTG), Prostaglandin H Synthase-1 (PTGS1) and PTGS2, PGF synthase (PTGFS), and PGE synthase (PTGES), PGF receptor (PTGFR), PGE receptors (PTGER2 and PTGER4), prostaglandin transporter (SLCO2A1) and hydroxyprostaglandin dehydrogenase-15 (HPGD). Endometrial biopsies were obtained from mares on day of ovulation (d0, n=4), late diestrus (LD, n=4), early luteolysis (EL, n=4) and after luteolysis (AL, n=4) during the cycle. Stages of the cycle were confirmed by plasma progesterone concentrations measured daily...
Expression of inflammation-associated genes in circulating leukocytes collected from horses with gastrointestinal tract disease. To investigate whether expression of inflammation-associated genes in leukocytes from horses with gastrointestinal tract (GIT) diseases correlated with the type of disease and outcome. Methods: 10 healthy horses and 50 horses with GIT disease. Methods: A blood sample was collected from each healthy horse or horse with GIT disease (during admission to the hospital). Leukocytes were isolated, diluted to a standard concentration, and frozen until RNA extraction. Expression of 14 genes associated with inflammation was quantified by use of a real-time quantitative reverse transcription PCR assay. R...
Expression of cytosolic phospholipase A2 in equine endometrium during the oestrous cycle and early pregnancy. Phospholipase A2 (PLA2) is a key enzyme for biosynthesis of PGF2α. Real-time RT-PCR and immunohistochemistry were used to determine transcription and cellular distribution of cytosolic PLA2 (cPLA2) in the equine endometrium during the oestrous cycle and early pregnancy. Endometrial biopsy and blood samples were collected from cycling mares on days (d) 8, 11, 15 and 18 (oestrus) (Day 0 = Day of ovulation; n = 5 for each day) and from pregnant mares (n = 4) on d15. Except for mares on d18 and some cyclic mares (n = 2) on d15 with low progesterone (P₄) concentrations (< 3.18 nm), P₄ conce...
Efficacy and safety of mitomycin C as an agent to treat corneal scarring in horses using an in vitro model. Mitomycin C (MMC) is used clinically to treat corneal scarring in human patients. We investigated the safety and efficacy of MMC to treat corneal scarring in horses by examining its effects at the early and late stages of disease using an in vitro model. Methods: An in vitro model of equine corneal fibroblast (ECF) developed was used. The ECF or myofibroblast cultures were produced by growing primary ECF in the presence or absence of transforming growth factor beta-1 (TGFbeta1) under serum-free conditions. The MMC dose for the equine cornea was defined with dose-dependent trypan blue exclusion...
Proinflammatory cytokine responses of cultured equine keratinocytes to bacterial pathogen-associated molecular pattern motifs. Further knowledge of equine keratinocyte physiology and keratinocyte response to various stimuli is important in developing a better understanding of disease states involving the epidermis. Objective: To assess the inflammatory cytokine response of cultured equine keratinocytes to various pathogen-associated molecular pattern molecules (PAMPs) from both Gram-negative and positive bacteria likely to be present in equine sepsis. Methods: Keratinocytes were isolated from skin of 2 horses and primary cultures performed. Keratinocytes were harvested for RNA extraction after exposure to lipopolysacc...
Bovine papillomavirus DNA can be detected in keratinocytes of equine sarcoid tumors. Bovine papillomavirus (BPV)-1 and -2 is linked to equine sarcoids, a commonly observed skin tumor in horses that is of considerable veterinary importance. Previous studies using in situ hybridization have detected BPV DNA only in fibroblasts and not in keratinocytes of sarcoids. In contrast, normal equine skin latently infected with BPV shows a dysplastic epithelium without dermal changes, similar to lesions induced by other papillomavirus types infecting the epithelium. The first goal of our study was to describe the epidermal and dermal characteristics of several stages in sarcoid developmen...
Subclinical infection and periodic shedding of equid herpesvirus 3. The temporary disruption of reproductive activities due to equine coital exanthema (ECE), caused by equid herpesvirus 3 (EHV-3), at thoroughbred breeding facilities and embryo transfer centres, has an appreciable economic impact. The aim of the present study was to estimate the prevalence of excretion of EHV-3 in mares without clinical symptoms under field conditions and the re-excretion patterns of the virus in two seropositive (presumably latently infected) mares maintained in isolation for 11 mo. The EHV-3 virus was detected in perineal-vaginal swabs by real time PCR in 14 (6%) of 220 thoro...
Effect of intravenous lidocaine administration on laminar inflammation in the black walnut extract model of laminitis. Laminitis is a serious complication of horses suffering from sepsis/endotoxaemia-related events. Laminitis in horses and organ injury in human sepsis are both reported to involve inflammatory injury to the laminae/organs including early activation of endothelium and leucocytes leading to emigration of neutrophils into the tissue interstitium. In the black walnut extract (BWE) model, systemic inflammatory events coincide with marked increase in laminar mRNA concentrations of inflammatory genes including proinflammatory cytokines (i.e. IL-1beta, IL-6), COX-2, chemokines (i.e. IL-8) and endotheli...
Sequence heterogeneity in the equi merozoite antigen gene (ema-1) of Theileria equi and development of an ema-1-specific TaqMan MGB assay for the detection of T. equi. Although a quantitative real-time PCR assay (qPCR) assay for the detection of Theileria equi has been developed and evaluated, it is possible that additional, as yet undetected 18S rRNA gene sequence variants may exist. A qPCR assay targeting a different gene, used in conjunction with the T. equi 18S rRNA qPCR assay, could assist in the detection of all T. equi genotypes in field samples. A T. equi ema-1-specific qPCR (Ueti et al., 2003) was tested on 107 South African field samples, 90 of which tested positive for T. equi antibody using the immuno-fluorescent antibody test (IFAT). The qPCR as...
An investigation into the suitability of a commercial real-time PCR assay to screen for Taylorella equigenitalis in routine prebreeding equine genital swabs. Standard bacteriological methods for identifying Taylorella equigenitalis in cervical smears are time consuming. Therefore, a more rapid real-time PCR assay was evaluated for its suitability in screening swabs. Objective: To compare the results of a commercially available real-time PCR assay with routine microbiological culture for the identification of T. equigenitalis, the causative organism of contagious equine metritis, in equine genital swab samples, under 'field trial' conditions. Methods: Routine prebreeding genital swabs (n=2072) collected from Thoroughbred mares and stallions during 2...
Fecal microbiota of horses in the clinical setting: potential effects of penicillin and general anesthesia. Antimicrobial treatment is associated with the spread of antimicrobial resistance and disturbances in the ecological balance of intestinal microbiota. In horses, the main adverse effect of antimicrobial treatment is colitis. We used culture and 16S rRNA gene based molecular methods to monitor the prevalence of antimicrobial resistance and changes in predominant fecal populations during penicillin treatment and general anesthesia of horses in the clinical setting. After 5 days of parenteral administration of penicillin, fecal Escherichia coli were resistant to multiple unrelated antimicrobial a...
Molecular characterization of glycogen synthase 1 and its tissue expression profile with type II hexokinase and muscle-type phosphofructokinase in horses. Muscle glycogen synthase (GYS1) is the rate-limiting enzyme in glycogen synthesis, and its activity is regulated by the phosphorylation states of certain amino acid residues encoded by the GYS1 gene. In the present study, the authors molecularly characterized the full-length equine GYS1 (eGYS1) cDNA and found that it contains a less common polyadenylation signal (AATACA). An amino acid alignment with other mammalian GYS1 showed that the phosphorylation sites in eGYS1 are completely conserved. Genomic DNA analysis revealed that the equine-specific substitutions (Glu 16 Asp and Ala 252 Thr) were...
Identification of differentially expressed genes associated with osteochondrosis in standardbred horses using RNA arbitrarily primed PCR. The aim of this study was to investigate genes for differential expression in cartilage of foals predisposed to osteochondrosis (OC). Tissue was sampled from the cranial part of the distal intermediate ridge of the tibia in the tarso-crural joint. Foals were considered predisposed to OC when parents had OC at the distal intermediate ridge of the tibia. RNA was isolated and subjected to arbitrarily primed PCR (RAP-PCR) followed by fingerprinting to screen for differentially expressed genes. By verification of results from the RAP-PCR fingerprint screening using real-time RT-PCR, we identified t...
Cytokine mRNA expressions after racing at a high altitude and at sea level in horses with exercise-induced pulmonary hemorrhage. To determine concentrations of cytokine mRNA in horses with exercise-induced pulmonary hemorrhage (EIPH) after racing. Methods: 97 Thoroughbreds. Methods: Following tracheobronchoscopy, the severity of EIPH was graded (scale of 0 to 4), and venous blood samples were collected from 10 horses in each grade. After RNA isolation and cDNA synthesis, real-time PCR assay was conducted to detect cytokinespecific mRNA for interleukin (IL)-1, IL-6, and IL-10; interferon (INF)-gamma; and tumor necrosis factor (TNF)-alpha. Results: Neither location nor grade of EIPH affected the expression of IL-1 and INF...
Transcriptional changes associated with recurrent airway obstruction in affected and unaffected horses. To identify differentially expressed genes in pulmonary tissues of horses affected with summer pasture-associated obstructive pulmonary disease (SPAOPD), which is a form of recurrent airway obstruction (RAO), compared with those of unaffected horses. Methods: 6 horses with SPAOPD-RAO and 6 unaffected (healthy) horses. Methods: Horses were assigned to 2 groups on the basis of medical history, clinical score, and transpleural pressure. Total RNA from each of the 5 lung lobes of each of the 6 SPAOPD-RAO-affected horses was extracted and pooled. Similarly, total RNA from unaffected horses was pool...
Development and optimisation of a duplex real-time reverse transcription quantitative PCR assay targeting the VP7 and NS2 genes of African horse sickness virus. Nucleotide sequences of 52 South African isolates of African horse sickness virus (AHSV) collected during 2004-2005 and including viruses of all nine AHSV serotypes, were used to design and develop a duplex real-time reverse transcription quantitative PCR (RT-PCR) assay targeting the VP7 (S8) and NS2 (S9) genes of AHSV. The assay was optimized for detection of AHSV in fresh and frozen blood of naturally infected horses. Assay performance was enhanced using random hexamers rather than gene-specific primers for RT, and with denaturation of double-stranded RNA in the presence of random hexamers. ...
Identification of Rhodococcus equi lipids recognized by host cytotoxic T lymphocytes. Immune adult horses have CD8(+) cytotoxic T lymphocytes (CTLs) that recognize and lyse Rhodococcus equi-infected cells in an equine lymphocyte alloantigen (ELA)-A [classical major histocompatibility complex (MHC) class I]-unrestricted fashion. As protein antigens are MHC class I-restricted, the lack of restriction suggests that the bacterial antigens being recognized by the host are not proteins. The goals of this study were to test the hypothesis that these CTLs recognize unique R. equi cell-wall lipids related to mycobacterial lipids. Initial experiments showed that treatment of soluble R. e...
Real-time RT-PCR for the detection and quantitative analysis of equine rhinitis viruses. Equine rhinitis viruses (ERV) cause respiratory disease and loss of performance in horses. It has been suggested that the economic significance of these viruses may have been underestimated due to insensitive methods of detection. Objective: To develop a sensitive, rapid, real-time RT-PCR (rRT-PCR) assay suitable for the routine diagnosis and epidemiological surveillance of the A and B variants of ERV. Methods: TaqMan primer probe sets for ERAV and ERBV were designed from conserved regions of the 5' UTR of the ERV genome. Over 400 samples from both clinically affected and asymptomatic horses w...
Effects of glucocorticoids and interleukin-1 beta on expression and activity of aggrecanases in equine chondrocytes. To determine effects of interleukin (IL)-1 beta and glucocorticoids on total glycosaminoglycan (GAG) loss and aggrecanase-mediated matrix degradation in equine cartilage. Methods: Cartilage from 24 equine cadavers free of sepsis and musculoskeletal disease. Methods: Effects of IL-1 beta, IL-1 beta with glucocorticoids (dexamethasone and triamcinolone, 10(-6) and 10(-7)M), and glucocorticoids alone on degradation of equine articular and nasal cartilage explants were assessed by measuring GAG release in media and GAG content in cartilage. Aggrecanase-mediated cleavage within the interglobular do...
Gene expression profiling from leukocytes of horses affected by osteochondrosis. Osteochondrosis (OC) is a developmental disease that affects growing horses and that severely affects their ability to perform. The genetic basis of its pathogenesis is poorly understood. The aim of the study was to analyze the transcript profile of leukocytes from horses affected with OC. Two transcriptome libraries were constructed from leukocytes of OC-affected and non-OC-affected horses using digital gene expression analysis (DGE) and real-time PCR. Statistical analysis allowed selection of 1,008 tags upregulated in the non-OC-affected group and 1,545 tags upregulated in the OC-affected gr...
Differential association of MUC5AC and CLCA1 expression in small cartilaginous airways of RAO-affected and control horses. Airway mucus accumulation is associated with indoor irritant and allergen exposure in horses with recurrent airway obstruction (RAO). Epidermal growth factor receptor (EGFR) and a chloride channel (calcium activated, family member 1; CLCA1) are key signalling molecules involved in mucin gene expression. Objective: We hypothesised that exposure to irritants and aeroallergens would lead to increased expression of the mucin gene eqMUC5AC and increased stored mucosubstance in the airways of RAO-affected horses, associated with increased neutrophils and CLCA1 and EGFR mRNA levels. Methods: We perfo...
