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.
Bhoora RV, Pienaar R, Cornelius F, Josemans A, Matthee O, Marumo R, Troskie C, Mans BJ.Quantitative real-time PCR assays previously developed for the detection of Theileria equi and Babesia caballi, were combined in a single multiplex TaqMan qPCR platform for the simultaneous detection of both heamoprotozoan parasites in equids. The multiplex equine piroplasmosis (M-EP) qPCR assay was shown to be efficient and specific. The detection limit was determined to be 1.4 × 10 % parasitized erythrocytes (PE) for T. equi and 2.8 × 10 % PE for B. caballi. The effect of differential DNA concentrations on the outcome of the M-EP qPCR for each target species was also investigated. ...
de Oliveira AM, Fonseca AA, Camargos MF, Orzil LM, Laguardia-Nascimento M, Oliveira AGG, Rodrigues JG, Sales ML, de Oliveira TFP, de Melo CB.Vesicular stomatitis is an infectious disease that occurs mainly in countries of the Western Hemisphere and affects cattle, swine and horses. The clinical symptoms in cattle and swine are similar to foot-and-mouth disease and include vesicular ulceration of the tongue and mouth. The disease requires a rapid and accurate differential diagnosis, aiming for immediate implementation of control measures. The objective of the present study was to develop and perform validation tests of multiplex RT-qPCR(s) for the detection of RNA from Alagoas vesiculovirus, considering the parameters of sensitivity...
Zoll WM, Prakoso D, Dark M, Liu J, Stockdale-Walden H, Long MT.Eosinophils within the central nervous system are abnormal and are usually associated with fungal or parasitic infections in horses. Causative agents include Halicephalobus gingivalis, Sarcocystis neurona, and Neospora hughesi. Confirmation of these organisms via specific testing is typically not performed, and final diagnoses are often presumptive. With molecular technology, many of these organisms can now be confirmed. This is important for emerging and zoonotic pathogens, including Angiostrongylus cantonensis, an emerging parasite of interest in the southeastern United States. We retrospect...
Lobanov VA, Peckle M, Massard CL, Brad Scandrett W, Gajadhar AA.Equine piroplasmosis (EP) is an economically significant infection of horses and other equine species caused by the tick-borne protozoa Theileria equi and Babesia caballi. The long-term carrier state in infected animals makes importation of such subclinical cases a major risk factor for the introduction of EP into non-enzootic areas. Regulatory testing for EP relies on screening of equines by serological methods. The definitive diagnosis of EP infection in individual animals will benefit from the availability of sensitive direct detection methods, for example, when used as confirmatory assays ...
D'Ascola A, Bruschetta G, Zanghì G, Campo S, Medica P, Campana S, Ferlazzo G, Gibbs BF, Ferlazzo AM.Serotonin (5-HT) is a neurohormone transported from plasma into platelets and leukocytes by a specific transporter (SERT). While it is known that the brain 5-HT system is modulated by physical exercise, the peripheral serotoninergic response to exercise is not yet fully elucidated. In particular, this study aimed to evaluate changes in plasma 5-HT levels and equine leukocyte SERT expression in response to treadmill exercise in untrained horses. Analyses were carried out pre- and post-treadmill exercise. 5-HT plasma levels were analysed by HPLC. Leukocytes and platelets were isolated to perform...
Qi T, Hu Y, Hu Z, Zhao S, Cullinane A, Lyons P, Gildea S, Wang X.Quantitation of virions is one of the important indexes in virological studies. To establish a sensitive and rapid quantitative detection method for equine arteritis virus (EAV), an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed by using two EAV nucleoprotein monoclonal antibodies (mAbs), 2B9 and 2B3, prepared in this study. After condition optimization, mAb 2B9 was used as the capture antibody, and HRP-labeled 2B3 was chosen as the detecting antibody. The AC-ELISA had a good standard curve when viral particles of the Bucyrus EAV strain were used as a reference stan...
Pusterla N, Leutenegger CM, Barnum SM, Byrne BA.In recent years, molecular approaches have been able to characterise the viability of equine upper respiratory tract pathogens using absolute molecular quantitation as well as detection of transcripts for virulence genes. Objective: The objective of this study was to investigate molecular surrogates for S. equi subspecies equi (S. equi) viability in biological samples from horses with strangles. Methods: Retrospective cross-sectional study. Methods: S. equi culture-positive and culture-negative upper airway secretions were assessed by qPCR at the genomic (gDNA) and complimentary DNA (cDNA) ...
Robles M, Nouveau E, Gautier C, Mendoza L, Dubois C, Dahirel M, Lagofun B, Aubrière MC, Lejeune JP, Caudron I, Guenon I, Viguié C, Wimel L....Obesity is a growing concern in horses. The effects of maternal obesity on maternal metabolism and low-grade inflammation during pregnancy, as well as offspring growth, metabolism, low-grade inflammation, testicular maturation and osteochondrotic lesions until 18 months of age were investigated. Twenty-four mares were used and separated into two groups at insemination according to body condition score (BCS): Normal (N, n = 10, BCS ≤4) and Obese (O, n = 14, BCS ≥4.25). BCS and plasma glucose, insulin, triglyceride, urea, non-esterified fatty acid, serum amyloid A (SAA), leptin and adiponect...
Pöschke A, Krähling B, Failing K, Staszyk C.The equine periodontal ligament (PDL) is a fibrous connective tissue that covers the intra-alveolar parts of the tooth and anchors it to the alveolar bone-it, therefore, provides a similar function to a tendinous structure. While several studies have considered the formation and structure of tendons, there is insufficient information particularly on the molecular composition of the PDL. Especially for the equine PDL, there is limited knowledge concerning the expression of genes commonly regarded as typical for tendon tissue. In this study, the gene expression of, e.g., (, and fibrocartilage m...
Kalpokas I, Mattos RC, Cavestany D, Martínez MN, Perdigón F, Meikle A.The effect of side of corpus luteum on uterine gene expression and protein localization of estrogen receptor α (ERα) and progesterone receptor (PR) in healthy cyclic and pregnant mares 13 days after ovulation (day 0) was investigated. Transcervical biopsies were performed to collect endometrium ipsilateral and contralateral regarding the side of corpus luteum on day 13 post-ovulation in cyclic (n = 6) and pregnant (n = 6) mares. Blood samples were collected daily from day 0 until the day of biopsy for 17β-estradiol (E2) and progesterone (P4) determinations. Receptor expression was d...
Tesena P, Korchunjit W, Taylor J, Wongtawan T.Gastric tissue biopsy and gene expression analysis are important tools for disease diagnosis and study of the physiology of the equine stomach. However, RNA extraction from gastric biopsy samples is a complex procedure because the samples contain low quantities of RNA and are contaminated with mucous protein and bacterial flora. The objectives of these studies were to compare the performance of RNA extraction methods and to investigate the sensitivity of commercial qPCR master mixes for gene expression analysis of gastric biopsy samples. Three commercial RNA extraction methods (TRIzol, GENEzol...
Maciel LFS, Silva ESM, Oliveira-Filho JP, Fritsch SC, Rossi RS, Lourenção JAC, Meira C.Administration of progesterone (P4) after estradiol is usually performed to prepare non-cyclic mares as embryo recipients. However, there are successful pregnancy reports after embryo transfer in non-cyclic mares treated only with progestins. The objective of this study was to evaluate endometrial gene expression and immunostaining for estrogen receptor alpha (ERα), beta (ERβ) and progesterone receptor (PR) in seasonal anestrous mares treated with long acting P4 (LA P4). Endometrial tissue from eight seasonal anestrous mares was collected immediately before administration of 1.5 g of LA P4 ...
Knych HK, Harrison L, Chouicha N, Kass PH.Intra-articular use of corticosteroids is commonplace in performance horses. Isoflupredone acetate (IPA) is one of four Food and Drug Administration approved corticosteroids for intra-articular use in horses. The lack of published reports describing the efficacy and duration of effects of this drug warrant further study. Objective: To assess the effects of intra-articular administration of IPA on the expression of selected anti- and pro-inflammatory and structural matrix genes following intra-articular administration to exercised Thoroughbred horses and to correlate these effects with drug con...
Peat FJ, Colbath AC, Bentsen LM, Goodrich LR, King MR.This study aimed to examine the influence of neodymium-doped yttrium aluminum garnet (Nd:YAG) laser irradiation on equine bone marrow-derived mesenchymal stem cell (MSC) viability, proliferation, and cytokine expression in vitro. Background: Photobiomodulation of cells using monochromatic light is a technique designed to influence cellular processes. Previous studies have shown dose-dependent effects of low-level laser irradiation on cell proliferation and cytokine expression in a range of cell types and species. Evidence for the influence of 1064 nm wavelength near-infrared irradiation on M...
Vieira FT, Acosta ICL, Martins TF, Filho JM, Krawczak FDS, Barbieri ARM, Egert L, Fernandes DR, Braga FR, Labruna MB, Dietze R.This work aims to identify and quantify the percentage of Rickettsia spp., Ehrlichia spp., Babesia spp. and Hepatozoon spp. positive pet dogs, and to identify ticks collected on these animals in the state of Espírito Santo, in the Southeast region of Brazil. The study included 378 dogs, 226 females and 152 males, of various breeds and ages (mean age of 4.1 years). All animals were examined for ticks, and whole blood was collected and processed by conventional PCR protocols for Babesia spp., Anaplasmataceae, Hepatozoon spp. and by real-time PCR for Ehrlichia canis. Of the 378 dogs examined, 15...
Tick-borne encephalitis (TBE) is a zoonotic flaviviral infection that is a growing public health concern in European countries. The aims of this research were to detect and characterize tick-borne encephalitis virus (TBEV) in Ixodes ricinus ticks at presumed natural foci in Serbia, and to determine seroprevalence of TBEV IgG antibodies in humans and animals. A total of 500 I. ricinus ticks were examined for the presence of TBEV by real-time RT-PCR, and conventional nested PCR and sequencing. To determine TBEV seroprevalence, 267 human sera samples were collected, as were 200 sera samples from ...
Miyata H, Itoh R, Sato F, Takebe N, Hada T, Tozaki T.Variants of the Myostatin gene have been shown to have an influence on muscle hypertrophy phenotypes in a wide range of mammalian species. Recently, a Thoroughbred horse with a C-Allele at the g.66493737C/T single-nucleotide polymorphism (SNP) has been reported to be suited to short-distance racing. In this study, we examined the effect of the Myostatin SNP on muscle fiber properties in young Thoroughbred horses during a training period. To investigate the effect of the Myostatin SNP on muscle fiber before training, several mRNA expressions were relatively quantified in biopsy samples from the...
Gim JA, Kim HS.Endogenous retroviruses (ERVs) have been integrated into vertebrate genomes and have momentously affected host organisms. Horses () have been domesticated and selected for elite racing ability over centuries. ERVs played an important role in the evolutionary diversification of the horse genome. In the present study, we identified six equine ERV families (EqERVs-E1, I1, M2, P1, S1, and Y4), their full-length viral open reading frames (ORFs), and elucidated their phylogenetic relationships. The divergence time of EqERV families assuming an evolutionary rate of 0.2%/Myr indicated that EqERV-S3 (7...
Carmona JU, Ríos DL, López C, Álvarez ME, Pérez JE.Platelet-rich plasma (PRP) preparations are used in horses with osteoarthritis (OA). However, some controversies remain regarding the ideal concentration of platelets and leukocytes to produce an adequate anti-inflammatory and anabolic response in the synovial membrane. The aims of this study were to study the influence of leukoconcentrated platelet-rich gel (Lc-PRG) and leukoreduced platelet-rich gel (Lr-PRG) supernatants on the quantitative expression of some proinflammatory and anabolic genes in equine synovial membrane explants (SMEs) challenged with lipopolysaccharide (LPS). SMEs from six...
Zarski LM, High EA, Nelli RK, Bolin SR, Williams KJ, Hussey G.Equine herpesvirus 5 (EHV-5) infection is associated with pulmonary fibrosis in horses, but further studies on EHV-5 persistence in equine cells are needed to fully understand viral and host contributions to disease pathogenesis. Our aim was to develop a quantitative PCR (qPCR) assay to measure EHV-5 viral copy number in equine cell cultures, blood lymphocytes, and nasal swabs of horses. Furthermore, we used a recently developed equine primary respiratory cell culture system to study EHV-5 pathogenesis at the respiratory tract. PCR primers and a probe were designed to target gene E11 of the EH...
Silva ESM, Ignácio FS, Fritsch SC, Zanoni DS, Pantoja JCF, Oliveira-Filho JP, Meira C.To test the hypothesis that the administration of 2.5 mg of estradiol benzoate (EB) followed by 1500 mg of long acting progesterone (LA P4) causes similar uterine changes and molecular dynamics in anovulatory mares to those observed in cyclic ones, we evaluated the changes of estrogen (ERα and ERβ) and progesterone receptors (PR) in anestrous, transitional and cyclic mares by RT-qPCR and immunohistochemistry. In addition, we evaluated uterine edema, tonus and estrogens and progesterone plasma profile. Endometrial biopsies were taken from anestrous and transitional mares immediately before ...
Cymerys J, Słońska A, Tucholska A, Golke A, Chmielewska A, Bańbura MW.Equine herpesvirus 1 (EHV-1), like other members of the Alphaherpesvirinae subfamily, is a neurotropic virus causing latent infections in the nervous system of the natural host. In the present study, we have investigated EHV-1 replication (wild-type Jan-E strain and Rac-H laboratory strain) during long-term infection and during the passages of the virus in cultured neurons. The studies were performed on primary murine neurons, which are an excellent in vitro model for studying neurotropism and neurovirulence of EHV-1. Using real-time cell growth analysis, we have demonstrated for the first tim...
Boyle AG, Stefanovski D, Rankin SC.Streptococcus equi subsp equi (S. equi) is the cause of "equine strangles" which is a highly infectious upper respiratory disease. Detection of S. equi is influenced by site of specimen collection, method of sampling, and type of diagnostic test that is performed. We hypothesized i) that a loop-mediated isothermal amplification (LAMP) assay that targets the S. equi-specific eqbE gene would be more sensitive than a realtime PCR assay that targets the S. equi-specific seeI gene and ii) that LAMP of specimens obtained by guttural pouch lavage (GPL) would be more sensitive than LAMP of nasopharyng...
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...
Cymerys J, Słońska A, Brzezicka J, Tucholska A, Chmielewska A, Rola J, Malik P, Bańbura MW.Equine herpesvirus type 1 (EHV-1) causes respiratory infections, abortion and neurological disorders in horses. Molecular epidemiology studies have demonstrated that a single-point mutation in DNA polymerase gene, resulting in an amino acid variation (N752/D752), is significantly associated with the neuropathogenic potential of EHV-1 strains. The aim of the study was to elucidate if there are any differences between neuropathogenic (EHV-1 26) and non-neuropathogenic (Jan-E and Rac-H) EHV-1 strains in their ability to infect neuronal cells. For the tested EHV-1 strains, cytopathic effect (CPE) ...
Kaspar A, Pfister K, Nielsen MK, Silaghi C, Fink H, Scheuerle MC.Strongylus vulgaris has become a rare parasite in Germany during the past 50 years due to the practice of frequent prophylactic anthelmintic therapy. To date, the emerging development of resistance in Cyathostominae and Parascaris spp. to numerous equine anthelmintics has changed deworming management and the frequency of anthelmintic usage. In this regard, reliable detection of parasitic infections, especially of the highly pathogenic S. vulgaris is essential. In the current study, two diagnostic methods for the detection of infections with S. vulgaris were compared and information on the occ...
Balasuriya UB, Lee PA, Tsai YL, Tsai CF, Shen YH, Chang HG, Skillman A, Wang HT, Pronost S, Zhang Y.Equine herpesvirus myeloencephalopathy (EHM), a major problem for the equine industry in the United States, is caused by equine herpesvirus-1 (EHV-1). In addition, EHV-1 is associated with upper respiratory disease, abortion, and chorioretinal lesions in horses. Here we describe the development and evaluation of an inexpensive, user-friendly insulated isothermal PCR (iiPCR) method targeting open reading 30 (ORF30) to detect both neuropathogenic and non-neuropathogenic strains on the field-deployable POCKIT™ device for point-of-need detection of EHV-1. The analytical sensitivity of the EHV-1 ...
Azarpeykan S, Dittmer KE.Housekeeping genes (HKGs) are used as internal controls for normalising and calculating the relative expression of target genes in RT-qPCR experiments. There is no unique universal HKG and HKGs vary among organisms and tissues, so this study aimed to determine the most stably expressed HKGs in the equine kidney. The evaluated HKGs included 18S ribosomal RNA (18S), 28S ribosomal RNA (28S), ribosomal protein L32 (RPL32), β-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase complex (SDHA), zeta polypeptide (YWHAZ), and hypoxanthine phosphoribosyltran...
Vina-Rodriguez A, Eiden M, Keller M, Hinrichs W, Groschup MH. (VEEV) is an from the family that causes epizootic outbreaks in equids and humans in Central and South America. So far, most studies use conventional reverse transcriptase PCR assays for the detection of the different VEEV subtypes. Here we describe the development of a TaqMan quantitative real-time reverse transcriptase PCR assay for the specific detection and quantitation of all VEEV subtypes which uses in parallel a universal equine encephalitis virus control RNA carrying target sequences of the three equine encephalitis viruses. The control RNA was used to generate standard curves for t...
Marth CD, Firestone SM, Glenton LY, Browning GF, Young ND, Krekeler N.Infectious endometritis is a major cause of reduced pregnancy rates in horses. The objectives of this study were to establish a timeline of the innate immune response in the uterus of healthy horses and to investigate the oestrous cycle effect on this. Endometrial biopsies were collected from five horses before and at 3, 12, 24, 48 and 72 h after inoculation of Escherichia coli, once in oestrus and once in dioestrus. They were analysed by quantitative real-time PCR, microbiology and histology. Neutrophil numbers increased from very low levels in the absence of inflammation to severe neutrophi...
Figueiredo MD, Moore JN, Vandenplas ML, Sun WC, Murray TF.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...
Pusterla N, Wilson WD, Conrad PA, Barr BC, Ferraro GL, Daft BM, Leutenegger CM.This study was designed to determine the relative levels of gene transcription of selected pathogens and cytokines in the brain and spinal cord of 12 horses with equine protozoal myeloencephalitis (EPM), 11 with equine herpesvirus type 1 (EHV-1) myeloencephalopathy, and 12 healthy control horses by applying a real time pcr to the formalin-fixed and paraffin-embedded tissues. Total rna was extracted from each tissue, transcribed to complementary dna (cDNA) and assayed for Sarcocystis neurona, Neospora hughesi, EHV-1, equine GAPDH (housekeeping gene), tumour necrosis factor (TNF)-alpha, interfer...
Lindbloom SM, Farmerie TA, Clay CM, Seidel GE, Carnevale EM.Human chorionic gonadotropin (hCG) was administered to mares in estrus with large, dominant ovarian follicles to initiate follicular and oocyte maturation. Follicular contents were collected at 0, 2, 4 and 6 h after hCG. Epiregulin, amphiregulin and phosphodiesterase (PDE) mRNA contents of granulosa cells (PDE 4D) were determined by reverse transcription and real-time PCR; PDE 3A mRNA content of single oocytes was determined similarly. Copy numbers of mRNA did not increase for PDE 3A or 4D over the time interval studied. Amounts of epiregulin and amphiregulin mRNA were correlated (r=0.98) when...
Jackson KA, Stott JL, Horohov DW, Watson JL.The objectives of this study were to quantify the induction of equine CD23 transcripts in equine peripheral blood mononuclear cells (PBMCs) and pulmonary alveolar macrophages cultured with recombinant equine IL-4 (rEq IL-4). PBMCs were isolated from blood drawn from four healthy horses. Bronchoalveolar lavage (BAL) fluid was collected from three healthy horses and alveolar macrophages were purified using adherence to plastic for 120 min. PBMCs and alveolar macrophages were cultured using four different conditions: rEq IL-4 and LPS, LPS alone, rEq IL-4 alone and a media control. Total RNA was i...
Wilson WC, Letchworth GJ, Jiménez C, Herrero MV, Navarro R, Paz P, Cornish TE, Smoliga G, Pauszek SJ, Dornak C, George M, Rodriguez LL.Sporadic outbreaks of vesicular stomatitis (VS) in the United States result in significant economic losses for the U.S. livestock industries because VS is a reportable disease that clinically mimics foot-and-mouth disease. Rapid and accurate differentiation of these 2 diseases is critical because their consequences and control strategies differ radically. The objective of the current study was to field validate a 1-tube multiplexed real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assay for the rapid detection of Vesicular stomatitis New Jersey virus and Vesicular st...
Miyata H, Itoh R, Sato F, Takebe N, Hada T, Tozaki T.Variants of the Myostatin gene have been shown to have an influence on muscle hypertrophy phenotypes in a wide range of mammalian species. Recently, a Thoroughbred horse with a C-Allele at the g.66493737C/T single-nucleotide polymorphism (SNP) has been reported to be suited to short-distance racing. In this study, we examined the effect of the Myostatin SNP on muscle fiber properties in young Thoroughbred horses during a training period. To investigate the effect of the Myostatin SNP on muscle fiber before training, several mRNA expressions were relatively quantified in biopsy samples from the...
Neil KM, Orth MW, Coussens PM, Chan PS, Caron JP.To determine whether glucosamine and chondroitin sulfate (CS) at concentrations approximating those achieved in plasma by oral administration would influence gene expression of selected mediators of osteoarthritis in cytokine-stimulated equine articular chondrocytes. Methods: Samples of grossly normal articular cartilage obtained from the metacarpophalangeal joint of 13 horses. Methods: Equine chondrocytes in pellet culture were stimulated with a subsaturating dose of recombinant equine interleukin (reIL)-1beta. Effects of prior incubation with glucosamine (2.5 to 10.0 microg/mL) and CS (5.0 t...
Ropka-Molik K, Stefaniuk-Szmukier M, Piórkowska K, Szmatoła T, Bugno-Poniewierska M.Apoptosis plays an important role in the regulation of healthy tissue growth and development as well as in controlling the maintenance of homeostasis in exercising muscles. During an intensive physical effort, the regulation of cell death by apoptosis results in the replacement of unaccustomed muscle cells by new cells that are better suited to exercise. The aim of this study was to determine the expression of two genes (SH3FR1 and SH3RF2) that control apoptosis in muscle tissues during training periods characterized by different intensities. The gene expression levels were estimated using rea...
Aleman M, Pickles KJ, Simonek G, Madigan JE.Trigeminal neuralgia or neuropathic pain has been regarded as a putative cause of idiopathic headshaking in horses. Equine herpesvirus-1 (EHV-1) infection and resultant postherpetic pain have been suggested as a possible cause of such neuropathic pain. Objective: To determine the presence of EHV-1 in the trigeminal ganglia of horses with idiopathic headshaking. Methods: Nineteen horses: control (n = 11, 9 geldings, 2 mares, median age 11 years) and headshaking (n = 8, all geldings, median age 11.5 years) horses were sourced from the equine research herd and caseload at the Veterinary Medical T...
Kolm G, Klein D, Knapp E, Watanabe K, Walter I.Lactoferrin (LF) is an estrogen-regulated glycoprotein with well-described antibacterial and immunomodulatory properties. The present study is the first report on LF expression in horse endometrial specimens. Mares chosen for the study were either resistant or susceptible for persisting mating-induced endometritis (PMIE) during the natural ovulatory cycle and in early pregnancy. Our investigations included immunostaining for LF protein and CD18, a leukocyte marker, as neutrophils are a possible source for LF in the endometrium. Quantification of LF mRNA was performed by use of real-time RT-PCR...
Kyaw-Tanner M, Pollitt CC.The dysadhesion and destruction of lamellar basement membrane of laminitis may be due to increased lamellar metalloproteinase activity. Characterising lamellar metalloproteinase-2 (MMP-2) and locating it in lamellar tissues may help determine if laminitis pathology is associated with increased MMP-2 transcription. Objective: To clone and sequence the cDNA encoding lamellar MMP-2, develop antibody and in situ hybridisation probes to locate lamellar MMP-2 and quantitate MMP-2 transcription in normal and laminitis tissue. Methods: Total RNA was isolated, fragmented by RT-PCR, cloned into vector a...
Buss DG, Sharma A, Giuliano EA, Mohan RR.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...
Sánchez-Matamoros A, Kukielka D, De las Heras AI, Sánchez-Vizcaíno JM.Cytokine secretion is one of the main mechanisms by which the immune system is regulated in response to pathogens. Therefore, the measurement of cytokine expression is fundamental to characterizing the immune response to infections. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) is widely used to measure cytokine mRNA levels, but assay conditions should be properly evaluated before analyzing important equine infections through relative quantification of gene expression. The aim of this study was to develop and evaluate a set of RT-qPCR assays for a panel of th...
Turan N, Yildirim F, Altan E, Sennazli G, Gurel A, Diallo I, Yilmaz H.The aim of the present study was to investigate abortion storms that occurred in the Marmara region of Turkey in 2008-2009 using a real-time PCR. Two aborted foetuses were necropsied and histo-pathological findings reported herein. Ten lungs, 3 brains and one nasal swab from 10 aborted foetuses, 6 nasal swabs and 3 vaginal swabs from aborting mares were included in this study. EHV-1 was isolated from the lung, liver and brain of 1 aborted foetus. EHV-1 DNA was detected in the lungs, livers and spleens of 2 necropsied foetuses and in 3 lungs from 10 foetuses submitted for diagnosis. A brain fro...
Pusterla N, Hussey SB, Mapes S, Leutenegger CM, Madigan JE, Ferraro GL, Wilson WD, Lunn DP.The objective of the current study was to compare the performance of 4 methods to quantify Equid herpesvirus 1 (EHV-1) by real-time polymerase chain reaction (PCR) in nasal secretions from experimentally and naturally infected horses. Nasal secretions were collected on the challenge day and daily thereafter for 13 days from 4 experimentally infected horses. Additional nasal swabs were collected from 30 horses with clinical signs consistent with natural EHV-1 infection. Absolute quantitation of EHV-1 target molecules was performed using standard curves for EHV-1 and equine glyceraldehyde-3-phos...
Phillips JC, Lembcke LM, Noltenius CE, Newman SJ, Blackford JT, Grosenbaugh DA, Leard AT.To determine the tissue-restricted expression pattern of tyrosinase mRNA in canine and equine melanocytic tumors and relative tyrosinase and major histocompatibility complex (MHC) I mRNA expression in variants of melanocytic tumors. Methods: 39 canine and 8 equine tumor samples and 10 canine and 6 equine normal tissue samples. Methods: RNA was isolated from formalin-fixed, paraffin-embedded tissues. Real-time PCR assays were designed to amplify canine and equine tyrosinase, S18 ribosomal RNA, and major histocompatibility complex I transcripts. Relative expression was determined by use of S18 a...
Gold JR, Perkins GA, Erb HN, Ainsworth DM.Septicemia initiates the production of pro-inflammatory (interleukin [IL] 1-beta [IL-1beta], interferon-gamma [IFN-gamma], IL-6), and anti-inflammatory (IL-4) cytokines. The transcription of some of these proteins (IL-8, IL-6) is linked to endotoxin-induced activation of the toll-like receptor 4 (TLR4) on peripheral blood mononuclear cells (PBMC). Objective: Septic foals fail to increase gene expression of IFN-gamma. Nonsurviving septic foals exhibit distinctive cytokine profiles. Methods: Twenty-one septic and 20 healthy neonatal foals. Methods: Using real-time polymerase chain reaction, gene...
Rocafort-Ferrer G, Leblond A, Joulié A, René-Martellet M, Sandoz A, Poux V, Pradier S, Barry S, Vial L, Legrand L.Equine piroplasmosis (EP) is a tick-borne disease caused by Babesia caballi and Theileria equi that is potentially emerging in non-endemic countries. We conducted a descriptive study to investigate EP prevalence and spatial distribution in an endemic region: the Camargue and the Plain of La Crau in France. In spring 2015 and 2016, we carried out sampling at stables (total n = 46) with a history of horses presenting chronic fever or weight loss. Overall, we collected blood from 632 horses, which were also inspected for ticks; these horses had been housed in the target stables for at least 1...
Cheung HW, Wong KS, Lin VYC, Wan TSM, Ho ENM.The misuse of genetic manipulation technology to enhance athletic performance is termed gene doping which is prohibited in human sports, horseracing, and equestrian sports. Although many qPCR assays have been developed, most assays employ genomic DNA (gDNA) from humans, non-human primates, and mice as a background and they may not be applicable for testing horse samples. This study aimed to develop a qPCR assay for the detection of human erythropoietin (hEPO) transgene in horse blood cells where the viral vectors used in gene therapy can reside for months. For the detection of hEPO transgene, ...
Zhang W, Cui S, Cheng XL, Wei F, Ma S.In this study, probe/primers of high specificity and sensitivity were selected to analyze donkey-hide gelatin for donkey DNA and to look for horse, ox, and pig DNA as possible adulterants. The mitochondrial CO I genes in donkey, horse, and ox were selected as target sequences for design and synthesis of three pairs of specific probes and primers. In addition, eight pairs of probe/primers were obtained via literature search. Out of these eleven groups of probe/primers, those with the highest specificity and sensitivity were selected, which was fulfilled by the screening firstly with animal hide...
Bhoora R, Buss P, Guthrie AJ, Penzhorn BL, Collins NE.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...
Jerele S, Davis E, Mapes S, Pusterla N, Navas González FJ, Iglesias Pastrana C, Abdelfattah EM, McLean A.Feral donkey removal from state land has raised concerns in terms of disease transmission between equine species. Disease outbreaks may occur as a result of the relocation of animals to new environments. Virus and bacteria DNA load and serum amyloid A derived from the pathogenic processes that they involve were measured in recently captured donkeys. Blood and nasal swabs were collected from 85 donkeys (Death Valley National Park, Shoshone, California); 24 were retested after 30/60 days in the Scenic (Arizona) long-term holding facility co-mingled with feral donkeys from Arizona and Utah. Quant...
Kinsley MA, Semevolos SA, Duesterdieck-Zellmer KF.The objective of this study was to elucidate gene and protein expression of Wnt signaling molecules in chondrocytes of foals having early osteochondrosis (OC) versus normal controls. The hypothesis was that increased expression of components of Wnt signaling pathway in osteochondral junction (OCJ) and cartilage canal (CC) chondrocytes would be found in early OC when compared to controls. Paraffin-embedded osteochondral samples (7 OC, 8 normal) and cDNA from whole cartilage (7 OC, 10 normal) and chondrocytes surrounding cartilage canals and osteochondral junctions captured with laser capture mi...
Bourebaba L, Bedjou F, Röcken M, Marycz K.Equine metabolic syndrome (EMS) refers to a cluster of associated abnormalities and metabolic disorders, including insulin resistance and adiposity. The numerous biological properties of mesenchymal stem cells (MSCs), including self-renewal and multipotency, have been the subject of many in-depth studies, for the management of EMS; however, it has been shown that this cell type may be affected by the condition, impairing thus seriously their therapeutic potential. Therefore, an attempt to rescue EMS adipose-derived stem cells (ASCs) with calystegines (polyhydroxylated alkaloids) that are endow...
Cappelli K, Felicetti M, Capomaccio S, Pieramati C, Silvestrelli M, Verini-Supplizi A.The stress response is a critical factor in the training of equine athletes; it is important for performance and for protection of the animal against physio-pathological disorders.In this study, the molecular mechanisms involved in the response to acute and strenuous exercise were investigated using peripheral blood mononuclear cells (PBMCs). Results: Quantitative real-time PCR (qRT-PCR) was used to detect modifications in transcription levels of the genes for matrix metalloproteinase-1 (MMP-1) and interleukin 8 (IL-8), which were derived from previous genome-wide expression analysis. Signific...
Azarpeykan S, Dittmer KE.Housekeeping genes (HKGs) are used as internal controls for normalising and calculating the relative expression of target genes in RT-qPCR experiments. There is no unique universal HKG and HKGs vary among organisms and tissues, so this study aimed to determine the most stably expressed HKGs in the equine kidney. The evaluated HKGs included 18S ribosomal RNA (18S), 28S ribosomal RNA (28S), ribosomal protein L32 (RPL32), β-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase complex (SDHA), zeta polypeptide (YWHAZ), and hypoxanthine phosphoribosyltran...
Szóstek AZ, Adamowski M, Galvão AM, Ferreira-Dias GM, Skarzynski DJ.Tumor necrosis factor-α (TNF) is a cytokine that plays important roles in functions of the endometrium. The aims of this study were to determine whether (i) ovarian steroids modulate TNF production by endometrial cells (Experiment 1); (ii) TNF effects on prostaglandin (PG) production in cultured equine endometrial cells and tissue (Experiment 2). Epithelial and stromal cells were isolated from equine endometrium (Days 2-5 of the estrous cycle; n=20) and treated after passage 1. In Experiment 1, epithelial and stromal cells were exposed to progesterone (P4; 10(-7)M), 17-β estradiol (E2; 10(-9...
Leise BS, Yin C, Pettigrew A, Belknap JK.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...
Silva LA, Klein C, Ealy AD, Sharp DC.This work examined how the conceptus modulates endometrial tissue remodeling and vascular development prior to implantation in mares. A macroscopic uterine examination was completed at day 21 of pregnancy. In situ morphology revealed that the endometrium involved in encroachment is restricted to the dorsal endometrium immediately overlying the yolk sac. The amount of stromal area occupied by blood vessels and the number of endometrial glands were increased during early pregnancy. Endometrial histomorphometry as well as the endometrial mRNA abundance and immunolocalization of VEGF, VEGFR1, VEGF...
Galvao AM, Ramilo DW, Skarzynski DJ, Lukasik K, Tramontano A, Mollo A, Mateus LM, Ferreira-Dias GM.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...
