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.
An T, Dugarjaviin M.(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...
Basso RM, Cerri FM, Braga PRC, Silva ROS, de Araújo Júnior JP, Oliveira-Filho JP, Pantoja JCF, Ribeiro MG, Arroyo LG, Borges AS.Diarrhea is a significant cause of morbidity and mortality in foals. Our hypothesis is that foals with diarrhea have a greater prevalence of ≥2 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 ...
Penzhorn L, Crafford JE, Guthrie AJ.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...
Ramsaran LN, Byron M, Parry S, Lection J, Back B, Grenier J, Cheong SH, Diel de Amorim M.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,...
Ketphan W, Sato M, Tsujimura K, Mizutani T, Takemae H.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...
McLachlan AD, Woolford L.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...
Parker JL, Berghaus LJ, Gordon J, Mathews L, Akinde MA, Hall DB, Peroni JF.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...
Tallmadge RL, Laverack M, Lejeune M, Crossley B, Diel DG.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) -Â targeting the equid genome. The EHV1-4MP assay analytical sensitiv...
Austin MMP, Ivey JLZ, Shepherd EA, Myer PR.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...
Beermann A, Hamza E, Reinhard S, Koch C, Oberhänsli T, Unger L.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 (median =â...
Djellouli M, Eddaikra N, Beneldjouzi A, Benikhlef R, Ghimire R, Wilkins M, Lafri I.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...
Zhou Y, Yao X, Meng J, Wang J, Zeng Y, Li L, Ren W.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...
Pipitpornsirikul P, Thangthamniyom N, Laikul A, Songkasupa T, Pathomsakulwong W, Apichaimongkonkun T, Kasemsuwan S, E-Kobon T, Lekcharoensuk P.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 naïve horses and 11 vaccinated horses during the 2020 outbreak. Whole blood was collected and subjected to RT real-time ...
Uprety T, Durazo J, Paul L, Metiner K, Ruby R, Loynachan A, Janes J, Kenndy L, Cassone L, Molly E, Quick M, Morgan J, Beyhan S, Erol E.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)...
Badr C, Arbi M, Souiai O, Larbi I, Nsiri J, Elbehi I, Bouslama Z, Bennour MA, Essaied MS, Ines Khosrof , Chabchoub A, Ben Aoun B, Ghram A, Lachheb J.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...
Iduu NV, Raiford D, Cohen ND, Landrock KK, Wang C.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...
Crisman EC, Furr M, Ramachandran A.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....
Hoxha I, Xhekaj B, Muja-Bajraktari N, Sekulin K, Unterköfler MS, Schlamadinger L, Situmorang T, Fuehrer HP, Obwaller AG, Camp JV, Walochnik J....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...
Mikaiel T, Waller A, Foote A, Cardwell JM, Mitchell J, Priestnall SL.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 97Â % DNA homology and respectively present serious equine health and welfare concerns. Currently there are few PCR assays targeting unique regions of...
Musoles-Cuenca B, Padilla-Blanco M, Vitale V, Lorenzo-Bermejo T, de la Cuesta-Torrado M, Ballester B, Maiques E, Rubio-Guerri C, Velloso Alvarez A.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...
Atay YE, Ekinci G, Öztürk AE, Timur MC, Mete A, Altınbay K, Derelli FM, Akar Y, Keleş İ.Equine Coital Exanthema (ECE) is an endemic herpesvirus disease primarily affecting the external genitalia and impairing mating activities in horses. Its extremely contagious nature, latency and subclinical features can result in outbreaks and significant economic losses. Transmission occurs primarily through mating activities; therefore, robust biosecurity measures are crucial in breeding facilities. This study aims to determine the clinical prevalence of ECE among horses in a covering station in Türkiye from 2021 to 2024. It also aims to assess the efficacy of routine PCR implementation wit...
Santiviparat S, Suthithanakom S, Bhanpattanakul S, Srisuwattanasagul S, Melde K, Stout TAE, Tharasanit T.Advances in endometrial tissue engineering have enabled the combination of modified scaffolding materials with modern cell culture technologies. Genipin and three-dimensional (3D) printing have advanced cell-tissue engineering by enabling the precise layering of cell-containing matrices while ensuring low cytotoxicity. This study aimed to advance equine endometrial tissue engineering by designing customized collagen scaffolds using 3D printing technology, while optimizing the genipin concentration to avoid toxicity. Genipin was tested at concentrations of 4, 2, 1, 0.5, 0.25, 0.125, and 0 mM on...
Bhoora RV, Mbaba TV, Troskie M, Ackermann RE, Collins NE.Theileria haneyi is an apicomplexan parasite closely related to Theileria equi, a known causative agent of equine piroplasmosis. The molecular distinction between these parasites relies on a nested polymerase chain reaction (PCR) assay, which has been reported to be unreliable. A recently reported indirect ELISA based on equi merozoite antigen 11 (Thema-11) of T. haneyi can detect geographically diverse T. haneyi strains. Since the ema-11 gene is exclusive to T. haneyi, it was chosen as the target for developing a TaqMan minor groove binder (MGBâ„¢) quantitative real-time PCR (qPCR). Published...
Cito F, Di Francesco CE, Averaimo D, Chiaverini A, Alessiani A, Di Domenico M, Cresci M, Rulli M, Cantelmi MC, Di Bernardo MD, Giammarino A.... subsp. (SEZ) is a major problem in equine veterinary medicine. Typically, a commensal in horses, SEZ can cause severe disease including respiratory infections, septicaemia and reproductive tract infections under certain conditions. Recent evidence suggests that humans can also develop severe disease infection through direct contact with infected animals or the consumption of contaminated unpasteurised milk and milk products. This study investigates SEZ strains isolated from nasal swabs of equidae in central Italy in 2023 to describe the epidemiology and genomic characteristics of circulating...
There are four genotypes of ovine papillomaviruses (OaPVs): OaPV1, OaPV2, and OaPV4, which are ovine delta papillomaviruses responsible for epithelial and mesenchymal cell infections, and OaPV3, an epitheliotropic Dyokappapapillomavirus associated with cutaneous tumors in sheep, including squamous cell carcinoma. Vaginal swabs of healthy mares were evaluated for the presence of PVs to investigate whether the vaginal virobiota of asymptomatic mares harbored OaPVs. High-performance digital droplet polymerase chain reaction (ddPCR) was used to quantitatively detect OaPV types 1-4 DNA in 94 vagina...
Cullinane A, Nelly M, Dayot L, Lukaseviciute G, Garvey M, Healy J, Gallagher R.This study describes a comparison of the detection of rotavirus in clinical samples from foals using two commercially available rapid antigen detection (RAD) kits, with the detection of rotavirus nucleic acid via a laboratory-based, in-house, real-time reverse transcription polymerase chain reaction (RT-PCR) assay. One hundred and forty freeze-thawed samples (70 that were RT-PCR-positive and 70 that were RT-PCR-negative on original tests) submitted to the diagnostic laboratory over a seven-year period were tested in addition to 123 fresh samples (15 RT-PCR-positive and 108 RT-PCR-negative) sub...
Tsujimura K, Bannai H, Kambayashi Y, Nemoto M, Ohta M.Equid alphaherpesvirus 1 (EqAHV1; Orthoherpesviridae, Varicellovirus equidalpha1) spreads by viremia to susceptible organs. Because EqAHV1 circulates in the bloodstream in a cell-associated manner, serum samples are not considered valuable for detecting EqAHV1 and have therefore not been tested by highly sensitive detection methods such as real-time PCR (rtPCR). We investigated whether EqAHV1 could be detected by this method in equine serum samples. We performed rtPCR on archived sera and peripheral blood mononuclear cells (PBMCs) collected from 3 horses experimentally inoculated with EqAHV1. ...
de Freitas MG, Dos Santos GH, Silva DLZ, Costa TD, Borges DGL, Reckziegel GH, Cleveland H, do Nascimento Ramos CA, Pereira FB, de Almeida Borges F.The strategic control of equine gastrointestinal nematodes is based on epidemiology. The aim is the reduction of the population of parasites as a whole in the hosts and environment while seeking the number of treatments that achieves the balance between not risking animal health and not exerting selection pressure for resistant parasites. This is the first study in a tropical region to assess a strategic control protocol for equines. The study was conducted in the municipality of Anastácio in the state of Mata Gross do Sul, Brazil, over a two-year period from April 2021 to March 2023, involvi...
Pusterla N, Lawton K, Barnum S, Vitomirov A, Anaya S, Naranatt P, Swadia H, Mendonsa E.The objective of this study was to evaluate a point-of-care (POC) PCR assay for the detection of Salmonella spp. in pooled environmental samples collected at an equine veterinary hospital. A total of 945 environmental samples were collected from high-risk areas, including ICU and isolation stalls, high-traffic areas, treatment rooms, and surgical suites. The environmental samples were collected using drag swabs placed in selenite broth and individually incubated at 35 °C for 20 h. Following the incubation period, 1 mL of up to 10 individual environmental samples were pooled together. Each poo...
Coles GC, Jackson F, Pomroy WE, Prichard RK, von Samson-Himmelstjerna G, Silvestre A, Taylor MA, Vercruysse J.Before revised World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines on the detection of anthelmintic resistance can be produced, validation of modified and new methods is required in laboratories in different parts of the world. There is a great need for improved methods of detection of anthelmintic resistance particularly for the detection of macrocyclic lactone resistance and for the detection of resistant nematodes in cattle. Therefore, revised and new methods are provided here for the detection of anthelmintic resistance in nematodes of ruminants, horses and ...
Kisiday JD, Kopesky PW, Evans CH, Grodzinsky AJ, McIlwraith CW, Frisbie DD.Bone marrow mesenchymal stem cells (BM-MSCs) and adipose-derived progenitor cells (ADPCs) are potential alternatives to autologous chondrocytes for cartilage resurfacing strategies. In this study, the chondrogenic potentials of these cell types were compared by quantifying neo-tissue synthesis and assaying gene expression and accumulation of extracellular matrix (ECM) components of cartilage. Adult equine progenitor cells encapsulated in agarose or self-assembling peptide hydrogels were cultured in the presence or absence of TGFbeta1 for 3 weeks. In BM-MSCs-seeded hydrogels, TGFbeta1 stimulate...
Cappelli K, Felicetti M, Capomaccio S, Spinsanti G, Silvestrelli M, Supplizi AV.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...
de la Fuente J, Ayoubi P, Blouin EF, Almazán C, Naranjo V, Kocan KM.Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae) causes human, equine and canine granulocytic anaplasmosis and tick-borne fever of ruminants. The rickettsia parasitizes granulocytes and bone marrow progenitor cells, and can be propagated in human promyelocytic and tick cell lines. In this study, microarrays of synthetic polynucleotides of 21,329 human genes were used to identify genes that are differentially expressed in HL-60 human promyelocytic cells in response to infection with A. phagocytophilum. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) of selec...
Slovis NM, Elam J, Estrada M, Leutenegger CM.Diarrhoea caused by infectious agents is common in foals but there is no comprehensive molecular work-up of the relative prevalence of common agents and appearance of coinfections. Objective: To determine the prevalence of 9 infectious agents in gastrointestinal (GI)-diseased and healthy foals with ages ranging from 1 to 20 weeks of age and to what degree coinfections are associated with clinical signs of GI disease. Methods: Retrospective controlled observational study. Methods: The population consisted of 88 Thoroughbred foals aged 2 days to 17 weeks born on 32 different studfarms in Kentuck...
da Silveira JC, Carnevale EM, Winger QA, Bouma GJ.Ovarian follicle growth and maturation requires extensive communication between follicular somatic cells and oocytes. Recently, intercellular cell communication was described involving cell-secreted vesicles called exosomes (50-150Â nm), which contain miRNAs and protein, and have been identified in ovarian follicular fluid. The goal of this study was to identify a possible role of exosomes in follicle maturation. Methods: Follicle contents were collected from mares at mid-estrous (~35Â mm, before induction of follicular maturation) and pre-ovulatory follicles (30-34Â h after induction of folli...
Haupt JL, Frisbie DD, McIlwraith CW, Robbins PD, Ghivizzani S, Evans CH, Nixon AJ.This study evaluated the potential of gene induced synoviocyte expression of a combination of insulin-like growth factor-I (AdIGF-I) and interleukin-1 receptor antagonist protein (AdIL-1Ra) to control articular cartilage degradation in vitro. Cartilage explants and synovial membrane were harvested from young mature horses. Synovial monolayers were established and either (1) maintained as untransduced controls; (2) transduced with AdIGF-I at 200 MOI in 500 microl serum-free medium; (3) transduced with AdIL-1Ra at 100 MOI; or (4) transduced with a combination of AdIGF-I (200 MOI) and AdIL-1Ra (1...
Szóstek-Mioduchowska AZ, Lukasik K, Skarzynski DJ, Okuda K.Transforming growth factor (TGF)-β1 not only regulates cell growth, development, and tissue remodeling, but it also participates in the pathogenesis of tissue fibrosis. In the equine endometrium, the concentration of TGF-β1 is correlated with endometrosis (equine endometrial fibrosis). In other tissues, TGF-β1 induces differentiation of many cell types into myofibroblasts. These cells are characterized by α-smooth muscle actin (α-SMA) expression and an ability to deposit excessive amounts of extracellular matrix (ECM) components. The aim of the study was to determine whether TGF-β1 plays...
Perkins GA, Goodman LB, Tsujimura K, Van de Walle GR, Kim SG, Dubovi EJ, Osterrieder N.A single nucleotide polymorphism in the equine herpesvirus 1 (EHV-1) DNA polymerase gene (ORF30 A(2254) to G) has been associated with clinical signs of equine herpes myeloencephalopathy (EHM). The purpose of our study was to determine the odds ratio for this genetic marker and EHM using a panel of field isolates from North America collected over the past twenty-three years. EHV-1 isolates cultured at the Cornell University Animal Health Diagnostic Laboratory from 1984 to 2007 were retrieved along with their clinical histories. DNA was extracted from these EHV-1 cultures and allelic discrimina...
Steinbusch MM, Fang Y, Milner PI, Clegg PD, Young DA, Welting TJ, Peffers MJ.The development of effective treatments for the age-related disease osteoarthritis and the ability to predict disease progression has been hampered by the lack of biomarkers able to demonstrate the course of the disease. Profiling the expression patterns of small nucleolar RNAs (snoRNAs) in joint ageing and OA may provide diagnostic biomarkers and therapeutic targets. This study determined expression patterns of snoRNAs in joint ageing and OA and examined them as potential biomarkers. Using SnoRNASeq and real-time quantitative PCR (qRT-PCR) we demonstrate snoRNA expression levels in murine age...
BÃ¥verud V, Johansson SK, Aspan A.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...
Alicka M, Kornicka-Garbowska K, Kucharczyk K, Kępska M, Rӧcken M, Marycz K.Progressive loss of cell functionality caused by an age-related impairment in cell metabolism concerns not only mature specialized cells but also its progenitors, which significantly reduces their regenerative potential. Adipose-derived stem cells (ASCs) are most commonly used in veterinary medicine as an alternative treatment option in ligaments and cartilage injuries, especially in case of high-value sport horses. Therefore, the main aim of this study was to identify the molecular alternations in ASCs derived from three age-matched horse groups: young ( 15 years old). ASCs were isolated ...
Bogaert L, Van Poucke M, De Baere C, Peelman L, Gasthuys F, Martens A.Real-time quantitative PCR can be a very powerful and accurate technique to examine gene transcription patterns in different biological conditions. One of the critical steps in comparing transcription profiles is accurate normalisation. In most of the studies published on real-time PCR in horses, normalisation occurred against only one reference gene, usually GAPDH or ACTB, without validation of its expression stability. This might result in unreliable conclusions, because it has been demonstrated that the expression levels of so called "housekeeping genes" may vary considerably in different t...
Janssen IJ, Krücken J, Demeler J, Basiaga M, Kornaś S, von Samson-Himmelstjerna G.Macrocyclic lactones (MLs) represent the major drug class for control of parasitic infections in humans and animals. However, recently reports of treatment failures became more frequent. In addition to human and ruminant parasitic nematodes this also is the case for the horse-nematode Parascaris equorum. Nevertheless, to date the molecular basis of ML resistance is still not understood. Unspecific resistance mechanisms involving transporters such as P-glycoproteins (Pgps) are expected to contribute to ML resistance in nematodes. Here, complete sequences of two P. equorum Pgps were cloned and i...
Sears WJ, Qvarnstrom Y, Dahlstrom E, Snook K, Kaluna L, Baláž V, Feckova B, Šlapeta J, Modry D, Jarvi S, Nutman TB.Angiostrongylus cantonensis (Ac), or the rat lungworm, is a major cause of eosinophilic meningitis. Humans are infected by ingesting the 3rd stage larvae from primary hosts, snails, and slugs, or paratenic hosts. The currently used molecular test is a qPCR assay targeting the ITS1 rDNA region (ITS1) of Ac. In silico design of a more sensitive qPCR assay was performed based on tandem repeats predicted to be the most abundant by the RepeatExplorer algorithm. Genomic DNA (gDNA) of Ac were used to determine the analytical sensitivity and specificity of the best primer/probe combination. This assay...
Kim CM, Blanco LB, Alhassan A, Iseki H, Yokoyama N, Xuan X, Igarashi I.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...
Radtke CL, Nino-Fong R, Esparza Gonzalez BP, Stryhn H, McDï¿® LA.To characterize equine muscle tissue- and periosteal tissue-derived cells as mesenchymal stem cells (MSCs) and assess their proliferation capacity and osteogenic potential in comparison with bone marrow- and adipose tissue-derived MSCs. Methods: Tissues from 10 equine cadavers. Methods: Cells were isolated from left semitendinosus muscle tissue, periosteal tissue from the distomedial aspect of the right tibia, bone marrow aspirates from the fourth and fifth sternebrae, and adipose tissue from the left subcutaneous region. Mesenchymal stem cells were characterized on the basis of morphology, ad...
Eivers SS, McGivney BA, Fonseca RG, MacHugh DE, Menson K, Park SD, Rivero JL, Taylor CT, Katz LM, Hill EW.Intense selection for elite racing performance in the Thoroughbred horse (Equus caballus) has resulted in a number of adaptive physiological phenotypes relevant to exercise; however, the underlying molecular mechanisms responsible for these characteristics are not well understood. Adaptive changes in mRNA expression in equine skeletal muscle were investigated by real-time qRT-PCR for a panel of candidate exercise-response genes following a standardized incremental-step treadmill exercise test in eight untrained Thoroughbred horses. Biopsy samples were obtained from the gluteus medius before, i...
Nielsen MK, Peterson DS, Monrad J, Thamsborg SM, Olsen SN, Kaplan RM.Strongylus vulgaris is an important strongyle nematode with high pathogenic potential infecting horses world-wide. Several decades of intensive anthelmintic use has virtually eliminated clinical disease caused by S. vulgaris, but has also caused high levels of anthelmintic resistance in equine small strongyle (cyathostomin) nematodes. Recommendations aimed at limiting the development of anthelmintic resistance by reducing treatment intensity raises a simultaneous demand for reliable and accurate diagnostic tools for detecting important parasitic pathogens. Presently, the only means available t...
Christoffersen M, Woodward E, Bojesen AM, Jacobsen S, Petersen MR, Troedsson MH, Lehn-Jensen H.The objective of the study was to evaluate the gene expression of inflammatory cytokines (interleukin [IL]-1β, IL-6, IL-8, IL-10, tumor necrosis factor [TNF]-α, IL-1 receptor antagonist [ra] and serum amyloid A (SAA) in endometrial tissue and circulating leukocytes in response to uterine inoculation of 105 colony forming units (CFU) Escherichia coli in mares. Before inoculation, mares were classified as resistant or susceptible to persistent endometritis based on their uterine inflammatory response to infusion of 109 killed spermatozoa and histological assessment of the endometrial quality. ...
HulÃnská D, Langrová K, Pejcoch M, Pavlásek I.The aim of this study was to detect Anaplasma phagocytophilum in wild and domesticated animals and to identify the phylogenetic relationships of different strains of this bacterium. We adapted six published conventional methods targeting 16S fragments for real-time polymerase chain reaction. Initial screening of samples from 419 animals found 37 Anaplasma positives, later confirmed with several different primers and a TaqMan probe. We also performed DNA quantification and melting curve analysis. The nucleic acid of Anaplasma sp. was detected in a higher percentage of cases in members of the de...
Cadby JA, Buehler E, Godbout C, van Weeren PR, Snedeker JG.The role of intrinsic and extrinsic healing in injured tendons is still debated. In this study, we characterized cell plasticity, proliferative capacity, and migration characteristics as proxy measures of healing potential in cells derived from the peritenon (extrinsic healing) and compared these to cells from the tendon core (intrinsic healing). Both cell populations were extracted from horse superficial digital flexor tendon and characterized for tenogenic and matrix remodeling markers as well as for rates of migration and replication. Furthermore, colony-forming unit assays, multipotency as...
Diallo IS, Hewitson G, Wright L, Rodwell BJ, Corney BG.Equid herpesvirus 1 (EHV1) is a major disease of equids worldwide causing considerable losses to the horse industry. A variety of techniques, including PCR have been used to diagnose EHV1. Some of these PCRs were used in combination with other techniques such as restriction enzyme analysis (REA) or hybridisation, making them cumbersome for routine diagnostic testing and increasing the chances of cross-contamination. Furthermore, they involve the use of suspected carcinogens such as ethidium bromide and ultraviolet light. In this paper, we describe a real-time PCR, which uses minor groove-bindi...
Altan E, Li Y, Sabino-Santos G, Sawaswong V, Barnum S, Pusterla N, Deng X, Delwart E.Metagenomics was used to identify viral sequences in the plasma and CSF (cerobrospinal fluid) of 13 horses with unexplained neurological signs and in the plasma and respiratory swabs of 14 horses with unexplained respiratory signs. Equine hepacivirus and two copiparvoviruses (horse parvovirus-CSF and a novel parvovirus) were detected in plasma from neurological cases. Plasma from horses with respiratory signs contained the same two copiparvoviruses plus equine pegivirus D and respiratory swabs contained equine herpes virus 2 and 5. Based on genetic distances the novel copiparvovirus qualified ...
Debrue M, Hamilton E, Joubert P, Lajoie-Kadoch S, Lavoie JP.Recent finding suggests that T-cells may be involved in the pathogenesis of heaves in horses. However, little is known concerning their possible contribution to pulmonary neutrophilia, a characteristic finding in heaves. Interleukin (IL)-17 is a cytokine secreted by activated T-cells that indirectly promotes the maturation, chemotaxis and activation of neutrophils. We therefore hypothesized that IL-17 may be involved in the recruitment of neutrophils into the airways and that its mRNA expression would be increased in bronchoalveolar lavage (BAL) cells of horses with heaves. Heaves susceptible ...
Bosch G, de Mos M, van Binsbergen R, van Schie HT, van de Lest CH, van Weeren PR.Extracorporeal shock wave therapy (ESWT) is frequently used in equine practice, but little is known about its biological action. Objective: To study the effects of ESWT on matrix structure and gene expression levels in normal, physiologically loaded tendinous structures in ponies. Methods: Six Shetland ponies, free of lameness and with ultrasonographically normal flexor and extensor tendons and suspensory ligaments (SL), were used. ESWT was applied at the origin of the suspensory ligament and the mid-metacarpal region of the superficial digital flexor tendon (SDFT) 6 weeks prior to sample taki...
Sales KG, Costa PL, de Morais RC, Otranto D, Brandão-Filho SP, Cavalcanti Mde P, Dantas-Torres F.Phlebotomine sand flies are blood-feeding insects of great medical and veterinary significance acting as vectors of Leishmania parasites. Studying the blood-feeding pattern of these insects may help in the understanding of their interactions with potential reservoir hosts of Leishmania parasites. In this study, we developed real time PCR assays for the identification of sand fly blood meal. Methods: Six pairs of primers were designed based on cytochrome b gene sequences available in GenBank of the following potential hosts: dog, cat, horse, chicken, black rat, and human. Firstly, SYBR Green-ba...
