Omneityยฎ 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.
Markers of respiratory inflammation in horses in relation to seasonal changes in air quality in a conventional racing stable. Airborne factors in a conventionally managed racing stable and markers of pulmonary inflammation in the stabled horses were investigated on 3 occasions at 6-month intervals, including 2 winter periods and the intervening summer period. The stable measurements included inside and outside ambient temperature and relative humidity, levels of total and respirable dust, endotoxin, and 1,3-beta-glucan. Horses (n = 12) were examined in detail clinically as well as by endoscopy, bronchoalveolar lavage (BAL) cytology, and real-time polymerase chain reaction (RT-PCR) analysis of BAL-cells for IL-6 and I...
Laminin receptor 1 is differentially expressed in thoracic and limb wounds in the horse. Healing of wounds located on the distal limbs of horses is often complicated by retarded epithelialization and the development of exuberant granulation tissue (proud flesh). Treatments that definitively resolve this pathological process are still unavailable. Molecular studies of the repair mechanism might contribute to the development of new therapeutic strategies. The study presented herein aimed to clone the full length cDNA and to study the spatio-temporal expression profile of mRNA and protein for LAMR1, previously attributed a role in wound epithelialization, during the repair of body an...
Real-time RT-PCR for detection of equine influenza and evaluation using samples from horses infected with A/equine/Sydney/2007 (H3N8). Equine influenza (EI) virus (H3N8) was identified in the Australian horse population for the first time in August 2007. The principal molecular diagnostic tool used for detection was a TaqMan real-time reverse transcription-polymerase chain reactions (RT-PCR) assay specific for the matrix (MA) gene of influenza virus type A (IVA). As this assay is not specific for EI, we developed a new EI H3-specific TaqMan assay targeting the haemagglutinin (HA) gene of all recent EI H3 strains. The IVA and the EI H3 TaqMan assays were assessed using in vitro transcribed RNA template, virus culture, diagnost...
Development of a real-time duplex TaqMan-PCR for the detection of Equine rhinitis A and B viruses in clinical specimens. Equine rhinitis A and B viruses (ERAV and ERBV) are respiratory viruses of horses belonging to the family Picornaviridae. Although these viruses are considered to cause respiratory disease in horses and are potentially infectious for humans, little is known about their prevalence and pathogenesis. Virus isolation is often unsuccessful due to their inefficient growth and lack of cytopathic effect in cell cultures. Therefore, molecular assays should be considered as the method of choice to detect infection in symptomatic or apparently healthy horses. In the present study, a novel real-time duple...
Cloning and expression of ADAM-related metalloproteases in equine laminitis. Equine laminitis is a debilitating disease affecting the digital laminae that suspend the distal phalanx within the hoof. While the clinical progression of the disease has been well documented, the molecular events associated with its pathogenesis remain largely unknown. Using real time quantitative PCR (RT-qPCR), we have investigated the expression of genes coding for proteins containing a Disintegrin and Metalloprotease domain (ADAM), as well as genes encoding the natural inhibitors of these enzymes (tissue inhibitor of metalloprotease; TIMP) in horses with naturally-acquired (acute, chronic...
Indices of inflammation in the lung and liver in the early stages of the black walnut extract model of equine laminitis. The liver and lung are not only described as "target organs" in sepsis in most species, but are purported to be sources of circulating inflammatory mediators central to the systemic inflammatory response syndrome (SIRS). As we have recently reported an inflammatory response in the laminar tissue in laminitis similar to that described in "target organs" in human sepsis, we investigated the inflammatory response of the lung and liver in the black walnut extract (BWE) model of equine laminitis to determine (1) if a similar systemic inflammatory response occurs in this laminitis model as described...
Differential gene expression associated with postnatal equine articular cartilage maturation. Articular cartilage undergoes an important maturation process from neonate to adult that is reflected by alterations in matrix protein organization and increased heterogeneity of chondrocyte morphology. In the horse, these changes are influenced by exercise during the first five months of postnatal life. Transcriptional profiling was used to evaluate changes in articular chondrocyte gene expression during postnatal growth and development. Methods: Total RNA was isolated from the articular cartilage of neonatal (0-10 days) and adult (4-5 years) horses, subjected to one round of linear RNA ampli...
Uterine involution and endometrial function in postpartum pony mares. To determine endometrial regeneration in postpartum mares by analysis of histologic features, apoptosis and cell proliferation markers, lectin binding, cytokines, and progesterone and estrogen receptors in endometrial biopsy specimens. Methods: 9 postpartum mares. Methods: Mares were examined on postpartum days 1, 9, and 16, and uterine biopsy specimens were obtained for histologic examination. Lectin binding was analyzed histochemically, and expressions of Ki-67 antigen (proliferation marker), lysozyme, and caspase 3 (apoptosis marker) were studied immunohistochemically. Gene expressions for ...
Detection of Lawsonia intracellularis by real-time PCR in the feces of free-living animals from equine farms with documented occurrence of equine proliferative enteropathy. The objective of this study was to determine whether Lawsonia intracellularis was present in the feces of free-living animals collected on two equine premises with documented occurrence of equine proliferative enteropathy (EPE). Fresh feces from black-tailed jackrabbits (Lepus californicus, n=100), striped skunks (Mephitis mephitis, n=22), feral cats (Felis catus, n=14), Brewer's Blackbirds (Euphagus cyanocephalus, n=10), Virginian opossums (Didelphis virginiana, n=9), raccoons (Procyon lotor, n=4), California ground squirrels (Spermophilus beecheyi, n=3), and coyotes (Canis latrans, n=2) were...
Role of bacteria in the pathogenesis of recurrent uveitis in horses from the southeastern United States. To determine the role of intraocular bacteria in the pathogenesis of equine recurrent uveitis (ERU) in horses from the southeastern United States by evaluating affected eyes of horses with ERU for bacterial DNA and intraocular production of antibodies against Leptospira spp. Methods: Aqueous humor, vitreous humor, and serum samples of 24 clinically normal horses, 52 horses with ERU, and 17 horses with ocular inflammation not associated with ERU (ie, non-ERU inflammation). Methods: Ribosomal RNA quantitative PCR (real-time PCR) assay was used to detect bacterial DNA in aqueous humor and vitreou...
Characterization of endothelin receptors in the peripheral lung tissues of horses unaffected and affected with recurrent airway obstruction. The purpose of the study was to determine and compare the expression of endothelin (ET) receptors in the peripheral lungs of healthy horses and those affected with recurrent airway obstruction (RAO) using reverse transcriptase polymerase chain reaction (RT-PCR), real-time PCR, Western blot analysis, and immunohistochemical techniques. Two groups of horses (7 healthy and 7 RAO-affected) were selected from a pool of horses destined for euthanasia. The grouping of horses was based on the history, clinical scoring, and pulmonary function testing. After euthanasia, gross postmortem evaluation of th...
Rapid and sensitive detection of African horse sickness virus by real-time PCR. A highly sensitive and specific TaqMan-MGB real-time RT-PCR assay has been developed and standardised for the detection of African horse sickness virus (AHSV). Primers and MGB probe specific for AHSV were selected within a highly conserved region of genome segment 7. The robustness and general application of the diagnostic method were verified by the detection of 12 AHSV isolates from all of the nine serotypes. The analytical sensitivity ranged from 0.001 to 0.15 TCID(50) per reaction, depending on the viral serotype. Real-time PCR performance was preliminarily assessed by analysing a panel of...
Detection of equine herpesvirus-1 in nasal swabs of horses by quantitative real-time PCR. Early identification of inhalation-transmitted equine herpesvirus type 1 (EHV-1) infections has been facilitated by the availability of a number of real-time quantitative PCR (qPCR) tests. A direct comparison between nasal swab qPCR and traditional virus isolation (VI) requires a method for normalizing the qPCR samples and controlling for PCR inhibitors present in some clinical samples. Objective: To quantify EHV-1 shedding in viral swabs using an internal control and to compare fast qPCR to VI for the detection of EHV-1 in nasal swabs from horses. Methods: Fifteen horses experimentally infect...
Equine herpesvirus infections in yearlings in South-East Queensland. Twelve nasal swabs were collected from yearling horses with respiratory distress and tested for equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4) by real-time PCR targeting the glycoprotein B gene. All samples were negative for EHV-1; however, 3 were positive for EHV-4. When these samples were tested for EHV-2 and EHV-5 by PCR, all samples were negative for EHV-2 and 11 were positive for EHV-5. All three samples that were positive for EHV-4 were also positive for EHV-5. These three samples gave a limited CPE in ED cells reminiscent of EHV-4 CPE. EHV-4 CPE was obvious after 3 days and...
Effects of the second-generation synthetic lipid A analogue E5564 on responses to endotoxin in [corrected] equine whole blood and monocytes. To evaluate proinflammatory effects of the second-generation synthetic lipid A analogue E5564 on equine whole blood and isolated monocytes and to determine the ability of E5564 to prevent LPS (lipopolysaccharide)-induced procoagulant activity (PCA); tumor necrosis factor (TNF)-alpha production; and mRNA expression of TNF-alpha, interleukin (IL)-1beta, IL-6, and IL-10 by equine monocytes. Methods: Venous blood samples obtained from 19 healthy horses. Methods: Whole blood and monocytes were incubated with Escherichia coli O111:B4 LPS, E5564, or E5564 plus E coli O111:B4 LPS. Whole blood and cell...
Exercise induced stress in horses: selection of the most stable reference genes for quantitative RT-PCR normalization. Adequate stress response is a critical factor during athlete horses' training and is central to our capacity to obtain better performances while safeguarding animal welfare. In order to investigate the molecular mechanisms underlying this process, several studies have been conducted that take advantage of microarray and quantitative real-time PCR (qRT-PCR) technologies to analyse the expression of candidate genes involved in the cellular stress response. Appropriate application of qRT-PCR, however, requires the use of reference genes whose level of expression is not affected by the test, by ge...
Real-time fluorogenic reverse transcription polymerase chain reaction assay for detection of African horse sickness virus. African horse sickness is an arthropod-borne disease of the equine included in the World Organization for Animal Health (OIE) list with important economic consequences for horse trade. The disease is caused by African horse sickness virus (AHSV; family Reoviridae, genus Orbivirus), which is transmitted by Culicoides midges. It is endemic in sub-Saharan Africa, spreading occasionally outside this area where the occurrence of Culicoides vectors allows virus transmission. Currently, only conventional (gel-based) reverse transcription polymerase chain reaction (RT-PCR) protocols are available for ...
Pathological, entomological, avian and meteorological investigation of a West Nile virus epidemic in a horse farm. Pathological, entomological and avian investigations were conducted during the summer of 2002, in a horse farm that had four cases of West Nile virus (WNV) infection in horses. All the four horses had encephalitis and WNV infection was confirmed by RT-PCR and in situ hybridization procedure. Forty-seven per cent of house sparrows that resided on the farm were tested positive for WNV infection. Mosquitoes (98%Culex pipiens) collected by trapping at the farm, during this period were positive for WNV. The meteorological data for year 2002 were compared to previous 16 years. The precipitation and ...
Semi-quantitative analysis of Ruminococcus flavefaciens, Fibrobacter succinogenes and Streptococcus bovis in the equine large intestine using real-time polymerase chain reaction. There is a need to further our understanding of the role that the equine hindgut ecosystem plays in digestive processes and diseases. The aim of the present study was to utilise the real-time PCR technique to determine the abundance of candidate cellulolytic (Ruminococcus flavefaciens; Fibrobacter succinogenes) and non-cellulolytic (Streptococcus bovis) bacteria in lumen contents from the caecum, ventral and dorsal colon, and rectum of healthy horses (n 14). Total DNA was extracted from frozen and lyophilised lumen contents, and PCR primers and Taqman probes were designed based on 16S rDNA seq...
Expression of histone 1 (H1) and testis-specific histone 1 (H1t) genes during stallion spermatogenesis. In eukaryotic cells, the major protein constituents of the chromatin are histones, which can be divided into five classes, identified as H1, H2A, H2B, H3 and H4. During normal spermatogenesis, a testis-specific H1t is expressed in primary spermatocytes and believed to facilitate histone to protamine exchanges during spermiogenesis. In equine testes we detected the H1 protein at 22kDa by western blot analysis while H1t was detected at 29kDa. H1 protein was found to be expressed in all germ cells up to elongating spermatids (Sc) at stage IV. In peripubertal animals, there was a prolonged express...
Comparison of two real-time reverse transcription polymerase chain reaction assays for the detection of Equine arteritis virus nucleic acid in equine semen and tissue culture fluid. Two previously developed TaqMan fluorogenic probe-based 1-tube real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assays (T1 and T2) were compared and validated for the detection of Equine arteritis virus (EAV) nucleic acid in equine semen and tissue culture fluid (TCF). The specificity and sensitivity of these 2 molecular-based assays were compared to traditional virus isolation (VI) in cell culture. The T1 real-time RT-PCR had a higher sensitivity (93.4%) than the T2 real-time RT-PCR (42.6%) for detection of EAV RNA in semen. However, the T1 real-time RT-PCR was les...
Technical note: a novel method for routine genotyping of horse coat color gene polymorphisms. The aim of this note is to describe a reliable, fast, and cost-effective real-time PCR method for routine genotyping of mutations responsible for most coat color variation in horses. The melanocortin-1 receptor, Agouti-signaling peptide, and membrane-associated transporter protein alleles were simultaneously determined using 2 PCR protocols. The assay described here is an alternative method for routine genotyping of a defined number of polymorphisms. Allelic variants are detected in real time and no post-PCR manipulations are required, therefore limiting costs and possible carryover contaminat...
Epithelial expression of mRNA and protein for IL-6, IL-10 and TNF-alpha in endobronchial biopsies in horses with recurrent airway obstruction. The aim of this study was to evaluate the contribution of bronchial epithelium to airway inflammation, with focus on mRNA and protein expression of cytokines of innate immunity IL-6, IL-10 and TNF-alpha, in horses with Recurrent Airway Obstruction (RAO) during exacerbation and in remission. Results: Despite marked clinical and physiologic alterations between exacerbation and after remission in the RAO horses no differences were detected in either cytokine mRNA or protein levels. Moreover, the expression of investigated cytokines in RAO horses on pasture did not differ from controls. In compari...
Distribution of the neurokinin-1 receptor in equine intestinal smooth muscle. Tachykinins have profound effects on equine intestinal motility, but the distribution of the neurokinin receptors (NKRs) through which they act is unknown. This study reports the distribution of one of these receptors, the neurokinin-1 receptor (NK1R), in smooth muscle throughout the equine intestinal tract. Objective: To quantify the distribution of the NK1R, based upon mRNA expression, in smooth muscle of different regions of the equine intestinal tract. Methods: Nine regions of the intestinal tract were sampled in 5 mature horses. Total RNA was isolated from smooth muscle and reverse transc...
Expression and localization of the mu-opioid receptor (MOR) in the equine cumulus-oocyte complex and its involvement in the seasonal regulation of oocyte meiotic competence. The micro-opioid receptor (MOR) was identified in equine oocytes, cumulus and granulosa cells. By RT-PCR, a 441bp fragment was observed. By immunoblotting, a 65 kDa band was detected in samples of winter anestrous whereas in cells recovered in breeding season, two bands, 65 and 50 kDa, were found. The 65 kDa band was significantly more intense in winter anestrous specimens. In samples recovered in the breeding season, this band significantly decreased with the raise of follicle size and was heavier in compact oocytes and cumulus cells. The protein was localized on the oolemma and within the cy...
Fatal pulmonary hemorrhage associated with RTX toxin producing Actinobacillus equuli subspecies haemolyticus infection in an adult horse. A case of fatal pulmonary hemorrhage in a 6-year-old American Paint mare with a 2-week history of intermittent coughing, fever, and epistaxis is described. Significant macroscopic abnormalities at postmortem examination were restricted to the respiratory system, and microscopically, severe pulmonary hemorrhage with suppurative bronchopneumonia was found. Actinobacillus equuli subsp. haemolyticus was cultured from a transtracheal wash performed antemortem as well as from the lungs at necropsy. The presence of airway-associated hemorrhage in conjunction with bacterial bronchopneumonia suggested ...
Equine laminitis: membrane type matrix metalloproteinase-1 (MMP-14) is involved in acute phase onset. Enzymatic separation at the hoof lamellar dermal-epidermal interface may play a role in the development of laminitis and characterising and locating matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of MMPs or TIMPs) in lamellar tissues may further understanding of pathogenesis. Objective: To clone and sequence the cDNA encoding lamellar MMP-14 and TIMP-2, and quantify their transcription in normal and laminitic tissue; and to develop antibody to locate MMP-14 in lamellar tissues. Methods: Tissue samples were obtained from an oligofructose induced model of laminitis. Tot...
Isolation of a gammaherpesvirus similar to asinine herpesvirus-2 (AHV-2) from a mule and a survey of mules and donkeys for AHV-2 infection by real-time PCR. Equids are commonly infected by herpesviruses, but isolation of herpesviruses from mules has apparently not been previously reported. Furthermore, the genomic relationships among the various equid herpesviruses are poorly characterized. We describe the isolation and preliminary characterization of a mule gammaherpesvirus tentatively identified as asinine herpesvirus-2 (AHV-2; also designated equid herpesvirus-7 (EHV-7)) from the nasal secretions (NS) of a healthy mule in northern California. The virus was initially identified by transmission electron microscopic examination of lysates of cell ...
Partial divergence of cytokine mRNA expression in bronchial tissues compared to bronchoalveolar lavage cells in horses with recurrent airway obstruction. The aim of this study was to investigate mRNA levels of cytokines in bronchial epithelium in horses with recurrent airway obstruction (RAO) during acute crisis and remission. Additionally, cytokine mRNA levels in endobronchial biopsies and bronchoalveolar lavage (BAL) cells were compared. Seven RAO horses were examined while in respiratory crisis following provocation and again while in remission after 2 months on pasture, during which time six healthy horses on pasture were also examined. Quantitative real-time PCR (RT-PCR) was used to assess mRNA expression for cytokines IL-5, IL-6, IL-8, IL...
