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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.
Differential Serum Cytokine Profiles in Aborting and Subclinically Infected Pregnant Mares with Equine Herpesvirus Type 1. Equine herpesvirus type 1 (EHV-1) is a major cause of reproductive loss in mares. This cross-sectional field study evaluated serum concentrations of interleukin-2 (IL-2), interleukin-10 (IL-10), interferon-alpha (IFN-ฮฑ), and interferon-gamma (IFN-ฮณ), together with the IL-10/IL-2 and IFN-ฮณ/IL-10 ratios, in 26 vaccinated pregnant Thoroughbred mares. The analytical cohort comprised healthy, EHV-1 PCR-negative mares (HM, n = 10), EHV-1 PCR-positive mares sampled within 4 h after fetal expulsion at approximately the sixth month of gestation (AM, n = 9), and EHV-1 PCR-positive pregnant mares with...
Alphavirus madariaga-associated encephalomyelitis in equines from the Brazilian Pantanal reveals arbovirus circulation in a peri-urban environment. Alphavirus madariaga (MADV), a member of the eastern equine encephalitis virus complex, is an emerging arbovirus in South America, with limited information regarding its occurrence and molecular characterization in equine populations. This study reports the detection and genetic characterization of MADV in equines from the state of Mato Grosso, Brazil. Two horses presenting acute neurological disease with a clinical course of approximately two days were evaluated. Histopathological examination revealed nonsuppurative meningoencephalomyelitis. Molecular screening for neurotropic viruses using T...
Identification of a Novel Slow-Gray STX17 Lineage in Japanese Thoroughbreds via a Multi-tiered Copy Number Analysis Workflow. Gray is a dominant coat color phenotype in horses caused by aโ~โ4.6ย kb tandem triplication within intron 6 of syntaxin 17 (STX17). The copy number variation (CNV) of the duplicated segment influences the graying rate. The rare G2 allele (CNVโ=โ2) is associated with a slower graying rate compared to the common G3 allele (CNVโ=โ3) and is also relevant to melanoma risk. Current assays are limited because long and accurate PCR (LA-PCR) detects only the presence or absence of duplications, while droplet digital PCR (ddPCR) cannot reliably distinguish certain genotypes such as G3/g and ...
Prevalence of Clostridioides difficile in hospitalized neonatal mare-foal pairs from Alberta and direct comparison of fecal culture with toxin gene detection. is an important pathogen in the feces of adult horses and foals. It can be clinically diagnosed using several testing methods, including culture isolation and, more commonly, direct toxin gene detection from feces using molecular methods. The importance of monitoring shedding in foals or dams during hospitalization remains unclear. The objectives of this study were to determine the prevalence of fecal shedding in mare-foal pairs and to compare detection using isolation and a commercial real-time polymerase chain reaction (qPCR) for toxin A and B genes in feces. Foals and their mares admitte...
Evaluation of two point-of-care molecular diagnostic platforms for rapid detection of equine Hendra virus. Hendra virus (HeV) is a lethal zoonotic pathogen endemic to eastern Australia, posing significant risks to equine and human health. Rapid field detection of HeV enables timely intervention and outbreak management. This study evaluated candidate point-of-care (POC) molecular diagnostic platforms for HeV detection in equine samples: including a loop-mediated isothermal amplification (DARQ RT-LAMP) assay and real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR). Comparative analytical evaluation demonstrated that RT-qPCR exhibited superior analytical sensitivity relativ...
miRNA Sequencing and Differential Analysis of Testis in 1-Year-Old and 2-Year-Old Kazakh Horses. The represents a significant genetic resource within China's population, characterized by notable resilience and an ability to thrive on coarse forage. Nevertheless, a decline in its numbers has been observed recently, making the improvement of its reproductive performance crucial for the preservation of this breed and the advancement of the related industry. In this study, testicular tissues from 1-year-old (pre-pubertal) and 2-year-old (post-pubertal) were analyzed. miRNA sequencing was conducted on tissues from these age groups, followed by bioinformatics analyses to elucidate the functi...
Comparative phenotypic and transcriptomic analysis of equine ovarian follicular aspirates-derived mesenchymal stromal cells and dermal fibroblasts. Mesenchymal stromal cells (MSCs) are widely used in human cell-based therapies and recent research is focused on the use of MSCs in equine regenerative medicine. Recently, MSCs have been isolated from equine follicular aspirates; however, a major concern during isolation is the potential contamination with fibroblasts, the predominant stromal cell type in many tissues. Furthermore, both cell types share the minimal criteria established by the International Society for Cellular Therapy (ISCT) for MSCs identification. Hence, our study aimed to compare the morphological characteristics, expansion...
Sex determination and genetic screening of equine embryos with whole genome amplification and real-time PCR. Reliable pre-implantation sex determination and genetic screening enables informed embryo transfer decisions in equine breeding while avoiding later interventions. We developed a streamlined workflow that couples rapid whole genome amplification (WGA) with a multiplex real-time PCR targeting ETSTY5 as a Y-specific marker and UBC as an autosomal control. On purified equine DNA, sex was correctly assigned down to 10 pg gDNA and to a single fibroblast cell. Direct testing of embryo biopsies without WGA yielded inconsistent results, whereas introducing a short WGA step produced tight allelic-discr...
Differential Expression of MITF, WNT3A, SLC7A11, and EDN3 in the Shoulder ‘Bider Marking’ of Dun Mongolian Horses. (1) Background: The "Bider marking" on the shoulder of the Dun Mongolian horse represents a unique pigmentation pattern, the molecular formation mechanism of which remains incompletely understood. This study investigates the differential expression and protein localization of pigment-related genes-specifically the core transcription factor , as well as , , and -in the skin. The analysis focuses on three distinct regions: the dark-colored area of the 'Bider marking' shoulder (BIDC), the light-colored area of the 'Bider marking' shoulder (BILC), and the non-Bider-marked shoulder area (NBIS). The...
Prevalence of potential enteric pathogens in diarrheic and non-diarrheic foals in Brazil. Diarrhea is a significant cause of morbidity and mortality in foals. Our hypothesis is that foals with diarrhea have a greater prevalence of โฅ2 organisms causing coinfections. We investigated the major microorganisms associated with diarrhea in 200 foals up to 1-y-old (100 diarrheic and 100 non-diarrheic). Fecal samples were analyzed by real-time PCR (rtPCR) for the detection of and toxin genes and , (genotyping for toxin-encoding genes), spp., , , , , , and spp. Rotavirus A and coronavirus were detected using reverse-transcription rtPCR. Fecal bacterial culture was also performed for ...
Direct single-nucleotide polymorphism genotyping from whole blood without DNA extraction. A subset of single-nucleotide polymorphisms (SNPs) are associated with various phenotypes, including diseases, coat colour, and athletic performance, and are widely used in medical and veterinary fields. In this study, we developed a novel method for direct SNP genotyping from whole blood without DNA extraction. Thoroughbred blood samples were diluted 100-fold with Milli-Q water and analysed using real-time polymerase chain reaction (PCR) with hydrolysis probes. Specificity and sensitivity were improved by increasing the annealing temperature and number of PCR cycles. Genotyping results for SN...
Enhancing African horse sickness virus detection: comparing and adapting PCR assays. African horse sickness (AHS) is the only equine disease for which the World Organisation for Animal Health (WOAH) gives official disease-free status, given that it poses a major threat to the equine industry. The disease is caused by AHS virus (AHSV; family , taxon species ), which is endemic in sub-Saharan Africa. Reverse-transcription quantitative real-time PCR (RT-qPCR) is a rapid, sensitive detection method used in the diagnosis of AHS and the certification of animals as negative for AHSV for the purpose of movement. Genetic variability of AHSV may influence the accuracy of RT-qPCR detecti...
Investigation of gene stability in equine luteal tissue during mid-diestrus phase and early pregnancy – Research Article. Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a technique that allows for the semi-quantification of mRNA transcripts present within a tissue of interest. Differences in the relative abundance of mRNA between samples detected by RT-qPCR require normalization with a reference gene or genes whose transcript abundance is stable within the tissue of interest independent of experimental conditions. In the field of equine reproductive studies, ACTB, GAPDH and B2M genes are the most widely used as reference genes for the normalization of RT-qPCR results. However,...
Identification of a novel equine rhinitis B virus detected in horse from Japan. Equine rhinitis B virus (ERBV), a member of the Picornaviridae family, is associated with mild to moderate respiratory illness in horses, yet its genomic diversity remains incompletely characterized. In this study, we performed metagenomic analysis on a rectal swab sample from a diarrheic foal confirmed to be positive for rotavirus A. Unexpectedly, we identified a highly divergent ERBV strain with a 9,448-nucleotide genome encoding a 2,721-amino-acid polyprotein. Sequence analysis revealed only 62.5-63.1% identity in the polyprotein and 47.1-49.8% in the VP1 region compared to known ERBV serot...
Equine abortion due to Chlamydia psittaci in South Australia with zoonotic transmission. Chlamydia psittaci was detected by real-time PCR in the lung, liver and kidney of an equine foetus that had aborted in South Australia in August 2023. The corresponding microscopic lesions included lymphocytic and histiocytic chorionitis, necrosis of placental villi associated with bacteria in the cytoplasm of trophoblastic epithelial cells, and multiple microgranulomas in the liver. Equine chlamydial abortion had not been diagnosed previously in South Australia. Eight days after examining the foetus and placenta, the veterinary pathologist developed fever and subsequently was admitted to hosp...
Impact of IL-6 and TGF-ฮฒ1 on equine mesenchymal stromal cell homing gene expression. Mesenchymal stromal cells (MSCs) support wound healing through immunomodulation and by promoting tissue organization. Cell tracking studies demonstrate that MSCs must 'home' to injuries in order to participate in reparative processes. It has been reported that in vivo cell tracking labels, including superparamagnetic iron oxide particles (SPIOs), can alter MSC homing. Homing is thought to occur in coordinated steps including tethering, activation, arrest, diapedesis, and migration, guided by chemotactic stimuli. Equine distal limb injuries, which are commonly treated with MSCs, possess a disti...
A multiplex real-time PCR assay for detection of equid herpesvirus 1 and 4. Equid herpesvirus (EHV) 1 and -4 are common viral pathogens of horses that can cause upper respiratory disease, neurological disease, abortion, and death. As characteristic alphaherpesviruses, both EHV-1 and EHV-4 can establish latency, resulting in a lifelong carrier state in infected animals. Here we describe the development and validation of a rapid and sensitive multiplex real-time PCR assay (EHV1-4MP) that simultaneously detects EHV-1 and EHV-4 and includes an endogenous internal control - melanocortin 1 receptor (MC1R) -ย targeting the equid genome. The EHV1-4MP assay analytical sensitiv...
Methodologies to Identify Metabolic Pathway Differences Between Emaciated and Moderately Conditioned Horses: A Review of Multiple Gene Expression Techniques. Starvation in horses presents critical welfare, economic, and management challenges with underlying molecular mechanisms of metabolic modification and recovery left poorly defined. Prolonged caloric deprivation induces significant systemic shifts in carbohydrate, protein, and lipid metabolism, reflected in coordinated changes in tissue-specific gene expression. This review synthesizes current knowledge on equine metabolic responses to starvation, emphasizing pathways found through RNA sequencing (RNA-seq) and real-time quantitative polymerase chain reaction (RT-qPCR) studies. Molecular investi...
Selected microRNAs as biomarkers in sarcoid-affected horses under immunotherapy with a mistletoe extract. We investigated microRNAs (miRNAs) as potential prognostic biomarkers for equine sarcoid (ES) disease. In a breed-, age-, and sex-matched case-controlled study involving 45 ES-affected and 15 control horses, we assessed the diagnostic, prognostic, and theragnostic value of 3 miRNAs (eca-miR-127, eca-miR-379, eca-miR-432) in horses treated with European mistletoe () extract versus placebo. Whole-blood miRNA concentrations were measured using reverse-transcription quantitative real-time PCR (RT-qPCR) at 3 different times. We found that eca-miR-432 expression was lower in ES-affected (medianโ=โ...
Serological, molecular, and epidemiological assessment of Leishmania spp. in equids within a hotspot endemic area for cutaneous leishmaniasis in southeastern Algeria. Cutaneous leishmaniosis remains a major public health concern in Algeria, predominantly affecting humans and dogs. However, the role of other animal hosts, particularly equids, in the transmission cycle remains underexplored. This cross-sectional study presents the first systematic investigation of Leishmania spp. exposure and infection in equids, along with associated environmental and host factors, from southeastern Algeria, El Oued Wilaya, the third most affected region for human cutaneous leishmaniasis in the country. A total of 122 equids from eight communes were clinically examined and t...
Mechanisms of Variation in Abdominal Adipose Color Among Male Kazakh Horses Through Non-Coding RNA Sequencing. The Kazakh horse is a highly valuable indigenous Chinese breed known for its use in both milk and meat production. However, the mechanisms underlying color variation in the abdominal adipose tissue of this breed remain poorly understood. In this study, the sequencing of non-coding RNAs (ncRNAs) was conducted on abdominal adipose tissue of different colors from Kazakh horses, with the aim of investigating the molecular mechanisms responsible for this variation. A total of 205 differentially expressed long non-coding RNAs (DELncRNAs) including , , and ; 52 differentially expressed microRNAs (DEm...
The Viremic Phase and Humoral Immune Response Against African Horse Sickness Virus That Emerged in Thailand in 2020. African horse sickness (AHS), a life-threatening disease caused by African horse sickness virus serotype 1 (AHSV-1), emerged in Thailand in February 2020 with 607 cases and a 93% fatality rate. The outbreak was mitigated by vector control and a live attenuated virus (LAV) vaccine. Information regarding viremia and immunity after infection and vaccination during outbreaks are essential for controlling disease transmission. This study evaluated these parameters in 15 infected naรฏve horses and 11 vaccinated horses during the 2020 outbreak. Whole blood was collected and subjected to RT real-time ...
Detection of Neorickettsia risticii in antemortem fecal and postmortem fetal samples, with genomic insights from complete genome sequencing of a strain recovered from an aborted equine fetus. Neorickettsia risticii (N. risticii) is an obligatory intracellular bacterium that causes Potomac horse fever (PHF), a disease clinically characterized by diarrhea, pyrexia, and laminitis in horses. Although sporadic reports of N. risticii infection have been linked to abortion in mares, a detailed retrospective study, including genomic analysis of the pathogen from an aborted fetus, has not been published. This study examined 546 fecal samples from clinically ill horses (January 1, 2017-December 31, 2024) and 833 colon samples from aborted equine fetuses (September 20, 2018-December 31, 2024)...
Tracing the 2021 equine influenza Outbreak: First characterization and phylogeographic analysis of H3N8 Florida clade 1 virus in Tunisia. Equine influenza (EI) is a highly contagious viral respiratory disease affecting equids, with the potential of causing widespread outbreaks across Europe, the Americas, Asia, and other regions of the world. In Tunisia, in the spring of 2021, an EI virus outbreak occurred in a farm housing purebred Arabian horses that exhibited respiratory signs. This outbreak led to a national lockdown on horse movements and the cancellation of equestrian events. A total of 432 serum samples, collected from 2018 to 2021, were tested using ELISA competition assay. In addition, 100 Nasal swabs were collected dur...
High-resolution melting curve FRET-qPCR rapidly distinguishes Streptococcus equi subsp. equi and zooepidemicus. We developed a fluorescence resonance energy transfer (FRET)-based real-time PCR (qPCR) assay coupled with high-resolution melting (HRM) analysis to differentiate subsp. (SEE) from subsp. (SEZ), two closely related equine pathogens with distinct clinical implications. The assay targets a region of the 23S rRNA gene containing single-nucleotide polymorphisms that differentiate the subspecies. By combining subspecies-specific probes with melting temperature () analysis, the assay accurately distinguished SEE, SEZ, and mixed infections without the need for post-PCR processing. The entire work...
First detection of equine hepacivirus RNA in Stomoxys calcitrans (Diptera, Muscidae) in eastern Austria. Equine hepacivirus (EqHV) from the family, has been detected in horses worldwide with a global RNA prevalence of up to 7.9%. While vertical transmission and iatrogenic transmission with infected blood products have been demonstrated for this virus, field infection rates suggest an additional horizontal transmission route. The aim of this study was to investigate the potential role of (Diptera, Muscidae) โ a hematophagous fly that is found in stables with ruminants and horses as preferred hostsโ in the transmission of EqHV RNA. From 2021 to 2022, were collected from three horse barns in ...
High seroprevalence of Francisella tularensis with low titers in Oklahoma equids from 2021-2023 suggests widespread exposure in the state’s equine population. To determine the seroprevalence of Francisella tularensis in horses in Oklahoma and to describe factors associated with seropositivity. Unassigned: In this observational study, 238 frozen equine serum samples were tested for F tularensis by microagglutination assay (MAT). Samples were collected from 36 of 77 Oklahoma counties from 2021 through 2023. Exclusion criteria included insufficient sample quantity, cross-reactivity to Brucella abortus, and county outside of Oklahoma. The Fisher exact test was used to compare the proportion of positive tests by year, river basin, and topographic region....
First Detection of West Nile Virus (WNV) Lineage 2 in Mosquitoes in the Republic of Kosovo. West Nile virus (WNV, family Flaviviridae) is the most geographically widespread arbovirus affecting humans. It circulates between wild birds and mosquitoes, while humans and horses are dead-end hosts. In recent years, several outbreaks have been reported from European countries, including the Balkan Peninsula. In the Republic of Kosovo, a southern Balkan country, data on WNV are scarce, and neither mosquito monitoring nor WNV surveillance is established. To address this gap, we aimed to assess a first monitoring approach that should set the basis and support future large-scale activities in t...
Novel primers for the detection and differentiation of S. zooepidemicus and S. equi by PCR. Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) is a commensal opportunistic bacterium associated with outbreaks of equine respiratory disease alongside a diverse range of diseases in various species. The closely related Streptococcus equi subspecies equi (S. equi) is the causative agent of 'strangles', the most frequently diagnosed contagious equine disease. Despite differing clinical signs, the two subspecies share approximately 97ย % DNA homology and respectively present serious equine health and welfare concerns. Currently there are few PCR assays targeting unique regions of...
First Molecular Evidence of Equine Herpesvirus Type 1 (EHV-1) in Ocular Swabs of Clinically Affected Horses. Equine Herpesvirus Type 1 (EHV-1) is a significant pathogen within the subfamily, causing respiratory disease, abortions, and, in severe cases, equine herpesvirus myeloencephalopathy (EHM). While nasal swabs and blood samples are commonly used for real-time polymerase chain reaction (RT-PCR) diagnosis, variability in viral shedding necessitates exploring additional sample types. This study reports the first molecular detection of EHV-1 in ocular swabs from naturally infected horses during an outbreak in the Valencian Community in 2023. Nasal and ocular swabs were collected from ten symptomati...
