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.
Söderlund R, Bölske G, Holst BS, Aspán A.Infection by Mycoplasma felis is associated with ocular and respiratory disease in cats and respiratory disease in horses. A correct diagnosis is beneficial since the use of specific antimycoplasmal treatment can lead to resolution. The objective of the present study was to develop a real-time polymerase chain reaction (PCR) method based on dual-labeled fluorogenic probe technology, targeting the gene encoding elongation factor Tu (tuf ), for the fast and specific detection of M. felis. Specificity was achieved by basing the assay design on partial sequencing of the tuf gene in strains and cli...
Hill EW, Eivers SS, McGivney BA, Fonseca RG, Gu J, Smith NA, Browne JA, MacHugh DE, Katz LM.The role of molecular signalling pathways in the phenotypic adaptation of skeletal muscle to different exercise stimuli in the Thoroughbred horse has not been reported previously. Objective: To examine CKM, COX4I1, COX4I2 and PDK4 gene expression following high intensity sprint and moderate intensity treadmill exercise stimuli in skeletal muscle of Thoroughbred horses. Methods: Two groups of trained 3-year-old Thoroughbred horses participated. Group A (n = 6 females, n = 3 males) participated in an incremental step test (moderate intensity) to fatigue or HR(max) on a Sato high speed treadmill ...
Liburt NR, Adams AA, Betancourt A, Horohov DW, McKeever KH.Studies have demonstrated increases in mRNA expression for inflammatory cytokines following exercise in horses and have suggested those markers of inflammation may play a role in delayed onset muscle soreness. However, measurement of mRNA expression in white blood cells is an indirect method. No studies to date have documented the cytokine response to exercise directly in muscle in horses. Objective: This study tested the hypothesis that exercise increases cytokine markers of inflammation in blood and muscle. Methods: Blood and muscle biopsies were obtained from 4 healthy, unfit Standardbred m...
Joubert P, Cordeau ME, Lavoie JP.Heaves in horses is characterized by lower airway neutrophilic inflammation, and reversible airflow obstruction. Pulmonary macrophages contribute to the inflammation observed in a number of human and animal pulmonary diseases, and it has been postulated that they are responsible for the neutrophilic inflammation present in heaves by the release of cytokines and chemokines. To test this hypothesis, the mRNA expression of TNF-α, IL-1β, IL-8, and MIP-2 by macrophages isolated from bronchoalveolar lavage cells was quantified using real-time RT-PCR in horses with heaves (n-6) and controls (n-6). ...
Hilton H, Nieto JE, Moore PF, Harmon FA, Naydan DK, Snyder JR.To determine expression of cyclooxygenase (COX) genes 1 and 2 (also called prostaglandin-endoperoxide synthases 1 and 2) and stability of housekeeping gene expression during low-flow ischemia and reperfusion in the jejunum of horses. Methods: 5 healthy adult horses. Methods: Horses were anesthetized, and two 30-cm segments of jejunum were surgically exteriorized. Blood flow was maintained at baseline (untreated) values in 1 (control) segment and was decreased to 20% of baseline (low-flow ischemia) for 75 minutes, followed by 75 minutes of reperfusion, in the other (experimental) segment. Biops...
Dunowska M, Howe L, Hanlon D, Stevenson M.The significance of infection with Equid herpesvirus 2 (EHV-2) remains unresolved, mainly due to its widespread distribution, and frequent isolation of the virus not only from diseased animals, but also from clinically normal horses. It has been suggested that EHV-2 exerts its effects on the host indirectly, through predisposition to secondary infections. The aim of this study was to determine kinetics of EHV-2 infection among foals and to investigate the role that EHV-2 may play in development of Rhodococcus equi pneumonia on one farm. Serial blood samples were collected from 43 foals over a ...
Atli MO, Guzeloglu A, Dinc DA.WNT signaling pathway plays important roles in reproductive events. Aims were to (1) determine presence of WNT genes and their antagonists in equine endometrium; and (2) to evaluate their expression profiles during early pregnancy. Endometrial biopsies were obtained from mares on day of ovulation (d0, n=4) and on days of 14 (P14, n=4), 18 (P18, n=4), 22 (P22, n=4) of early pregnancy. Biopsies were also collected from cyclic mares during late diestrus (LD, on day of 13.5-14, n=4) and after luteolysis in estrus phase (AL, on day of 17.5-18, n=4) of the cycle. PCR was used to detect expression of...
Zhang L, Franchini M, Wehrli Eser M, Dip R.Accumulation of extracellular adenosine has been closely associated with human asthmatic responses. However, the relevance of adenosine signalling in equine airways has not previously been investigated. Objective: To determine the expression of adenosine receptors (AR) in bronchoalveolar lavage (BAL) cells and assess the reactivity of these cells to AR ligands ex vivo, employing IL-6 as readout of adenosinergic inflammatory signalling. Methods: Eight horses with varying degrees of lower airway inflammation and 10 healthy controls were analysed. Expression of AR-subtypes in each BAL sample was ...
Cook VL, Holcombe SJ, Gandy JC, Corl CM, Sordillo LM.Monocytes are among the initial cells that interact with circulating LPS. Binding of LPS to monocyte surface receptors triggers an intracellular signaling cascade and results in the production of proinflammatory cytokines. Ethyl pyruvate, a stable derivative of pyruvate, has been effective in mitigating LPS induced alterations in isolated human monocytes. We hypothesized that ethyl pyruvate would suppress proinflammatory gene expression in LPS-stimulated equine monocytes without affecting cell viability. Equine monocytes were isolated from whole blood using a sediment-gradient centrifugation p...
Kayis SA, Atli MO, Kurar E, Bozkaya F, Semacan A, Aslan S, Guzeloglu A.The aim was an evaluation of a set of housekeeping genes (HKGs) to be used in the normalization of gene expression in the equine endometrium. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine ribosyl transferase 1 (HPRT1), ubiquitin B (UBB), tubulin alpha 1 (TUBA1), ribosomal protein L32 (RPL32), beta-2-microglobulin (B2M), 18S rRNA (18S), and 28S rRNA (28S) HKGs were evaluated using real-time PCR and were compared in different physiological stages of the endometrium. Endometrial biopsies were obtained from mares on day of ovulation (d0, n=4), at late diestrus (LD, n=4), after lut...
Almeida J, Conley AJ, Mathewson L, Ball BA.In the mammalian testis, Leydig cells are primarily responsible for steroidogenesis. In adult stallions, the major endocrine products of Leydig cells include testosterone and estrogens. 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4)-isomerase (3βHSD) and 17α-hydroxylase/17,20-lyase (P450c17) are two key steroidogenic enzymes that regulate testosterone synthesis. Androgens produced by P450c17 serve as substrate for estrogen synthesis. The aim of this study was to investigate localization of the steroidogenic enzymes P450c17, 3βHSD, and P450arom and to determine changes in expression during dev...
Lavoie JP, Cesarini C, Lavoie-Lamoureux A, Moran K, Lutz S, Picandet V, Jean D, Marcoux M.There is limited information relating bronchoalveolar lavage (BAL) cytology and cytokine messenger ribonucleic acid (mRNA) expression in racehorses with inflammatory airway disease (IAD). HYPOTHESIS AND OBJECTIVE: We hypothesize that cytokine expression in BAL cells would correlate with cytology. Thus, we evaluated the mRNA expression of selected cytokines in BAL cells in racehorses with exercise intolerance and lower airway inflammation. Methods: Thirty-one client-owned Standardbred racehorses with exercise intolerance. Methods: Prospective, observational study. Cells were obtained by BAL, an...
Beekman L, Tohver T, Dardari R, Léguillette R.The stability of reference genes has a tremendous effect on the results of relative quantification of genes expression by quantitative polymerase chain reaction. Equine Inflammatory Airway Disease (IAD) is a common condition often treated with corticosteroids. The diagnosis of IAD is based on clinical signs and bronchoalveolar lavage (BAL) fluid cytology. The aim of this study was to identify reference genes with the most stable mRNA expression in the BAL cells of horses with IAD irrespective of corticosteroids treatment. Results: The expression stability of seven candidate reference genes (B2...
Paris DB, Kuijk EW, Roelen BA, Stout TA.Real-time quantitative PCR (qPCR) is invaluable for investigating changes in gene expression during early development, since it can be performed on the limited quantities of mRNA contained in individual embryos. However, the reliability of this method depends on the use of validated stably expressed reference genes for accurate data normalisation. The aim of the present study was to identify and validate a set of reference genes suitable for studying gene expression during equine embryo development. The stable expression of four carefully selected reference genes and one developmentally regula...
Klein C, Troedsson MH.Establishment and maintenance of pregnancy are critically dependent on embryo-maternal communication during the preimplantation period. The horse is one of the few domestic species in which the conceptus-derived pregnancy recognition signal has not been identified. To gain new insights into the factors released by the equine conceptus, transcriptional profiling analyses of conceptuses retrieved 8, 10, 12, and 14 days after ovulation were performed using a whole-genome microarray. Selected array data were confirmed using quantitative PCR, and the expression of proteins of interest was confirmed...
Leise BS, Faleiros RR, Watts M, Johnson PJ, Black SJ, Belknap JK.There is a need to assess the laminar inflammatory response in a laminitis model that more closely resembles clinical cases of sepsis-related laminitis than the black walnut extract (BWE) model. Objective: To determine if a similar pattern of laminar inflammation, characterised by proinflammatory cytokine expression, occurs in the CHO model of laminitis as has been previously reported for the BWE model. Methods: Sixteen horses administered 17.6 g of starch (85% corn starch/15% wood flour)/kg bwt via nasogastric (NG) tube were anaesthetised either after developing a temperature>38.9°C (DEV ...
Heimann M, Janda J, Sigurdardottir OG, Svansson V, Klukowska J, von Tscharner C, Doherr M, Broström H, Andersson LS, Einarsson S, Marti E....Equine insect bite hypersensitivity (IBH) is a seasonally recurrent, pruritic skin disorder caused by an IgE-mediated reaction to salivary proteins of biting flies, predominantly of the genus Culicoides. The aim of this study was to define T cell subsets and cytokine profile in the skin of IBH-affected Icelandic horses with particular focus on the balance between T helper (Th) 1, Th2 and T regulatory (Treg) cells. Distribution and number of CD4+, CD8+ and Forkhead box P3 (FoxP3)+ T cells were characterized by immunohistochemical staining in lesional and non-lesional skin of moderately and seve...
Hughes KJ, Nicolson L, Da Costa N, Franklin SH, Allen KJ, Dunham SP.Inflammatory airway disease (IAD) is a common disorder of performance horses and is associated with poor performance and accumulation of mucus and inflammatory cells in lower airway secretions. Horses with IAD frequently have increased relative counts of neutrophils in bronchoalveolar lavage fluid (BALF); less commonly relative counts of eosinophils and/or mast cells may be increased. The aetiopathogenesis of IAD is unknown and may involve innate and/or acquired immune responses to various factors including respirable dust constituents, micro-organisms, noxious gases and unconditioned air. The...
Song JW, Na KS, Tae SH, Kim YK.During the last two decades, Borna disease virus (BDV) has received much attention as a possible zoonotic agent, particularly as a cause of psychiatric disease. Although several studies have shown that BDV is present in Asia, BDV has not been detected in Korea. This study was designed to further investigate the presence of BDV infection in Korea. Methods: Blood samples were taken from 39 race horses and 48 jockeys. Antibody to BDV was detected by indirect immunofluorescence antibody test and RNA of BDV by real time reverse transcriptase PCR (rRT-PCR). Results: No evidence of BDV was detected i...
Brooks SA, Bailey E.Inflammatory damage to the digital laminae, a structure responsible for suspension of the distal skeleton within the hoof capsule, results in a painful and often life-threatening disease in horses called laminitis. There can be many diverse causes of laminitis; however, previous work in the horse has suggested that in each case, the inflammation and resulting tissue damage is consistent with the action of mediators released from mast cells (MC), as well as the downstream consequences of their activation. The recent development of molecular genetics tools to characterize cells based on their tr...
Capomaccio S, Cappelli K, Barrey E, Felicetti M, Silvestrelli M, Verini-Supplizi A.It is known that moderate physical activity may have beneficial effects on health, whereas strenuous effort induces a state resembling inflammation. The molecular mechanisms underlying the cellular response to exercise remain unclear, although it is clear that the immune system plays a key role. It has been hypothesized that the physio-pathological condition that develops in athletes subjected to heavy training is caused by derangement of cellular immune regulation. The purpose of the present study was to obtain information on endurance horse gene transcription under strenuous conditions and t...
Webb TL, Burnett RC, Avery AC, Olver CS.Characterization of anemia in horses presents a challenge, as they do not release reticulocytes into peripheral blood. Transferrin receptor (TfR) expression is highest on erythroid cells in people and rats, and measurement of a soluble serum form (sTfR) is used to quantify erythropoiesis in these species. We hypothesized that equine TfR (eTfR) expression is similar in quantity and distribution to that in these other species and thus has potential for characterization of the regenerative response in anemic horses. Objective: This study was conducted to clone and sequence the eTfR gene and measu...
Magdesian KG, Leutenegger CM.Clostridium difficile infection can occur in the dams of sick foals, but it is unknown if mares and foals share the same isolates. In this study, C. difficile isolates from fecal samples of 11 mares paired with 11 foals were genotyped by arbitrarily primed PCR; two mares and three foals in five mare-foal pairs had diarrhea. Fecal immunoassays were utilized to detect C. difficile common antigen and toxin A. Quantitative real-time PCR (qPCR) systems were developed to detect genes for toxins A and B, as well as for binary toxin B. Sequences of all toxins were present in all isolates, although onl...
Slough TL, Rispoli LA, Carnevale EM, Niswender GD, Bruemmer JE.A biopsy procedure was developed to enable repeated sampling of a single equine corpus luteum (CL) over the course of an estrous cycle. The tissue collected was utilized in characterizing mRNA abundance for genes involved in luteal formation, function, and regression in the cyclic mare. Serial biopsies of CL in cyclic mares (2.7 to 27.5 mg per biopsy) were collected using an ultrasound-guided transvaginal technique. Biopsies were collected from each mare on d 2 and 5 (d 0 = ovulation) of the estrous cycle, and every other day from d 12 through luteolysis. Samples were obtained from 4 mares wit...
Pronost S, Pitel PH, Miszczak F, Legrand L, Marcillaud-Pitel C, Hamon M, Tapprest J, Balasuriya UB, Freymuth F, Fortier G.The vast majority of equine arteritis virus (EAV) infections are inapparent or relatively mild, but may occasionally cause outbreaks of equine viral arteritis. The event observed in France during the summer of 2007 was the most important seen in the country, with mortality and disruption of economic activity. Objective: To describe the different stages seen during the outbreak and to show how molecular tools were used for both the detection and management of the crisis. Methods: EAV detection was performed by real-time reverse transcription-polymerase chain reaction (RT-PCR) in blood, nasal sw...
Peffers MJ, Milner PI, Tew SR, Clegg PD.SOX9 is a transcription factor that is essential for cartilage extracellular matrix (ECM) formation. Osteoarthritis (OA) is characterised by a loss of cartilage ECM. In chondrocytes SOX9 gene expression is regulated by osmotic loading. Here we characterise SOX9 mRNA regulation through static and cyclical application of hyperosmotic conditions in normal and OA monolayer equine chondrocytes. Furthermore, we investigate whether extracellular signal-regulated protein kinase (ERK)1/2 mitogen-activated protein kinases (MAPK) pathways have a role in this regulation of SOX9. Methods: Equine chondrocyt...
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...
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...
Atli MO, Kurar E, Kayis SA, Aslan S, Semacan A, Celik S, Guzeloglu A.The aim was to evaluate expression of genes involved in the biosynthesis of prostaglandins (PTG), Prostaglandin H Synthase-1 (PTGS1) and PTGS2, PGF synthase (PTGFS), and PGE synthase (PTGES), PGF receptor (PTGFR), PGE receptors (PTGER2 and PTGER4), prostaglandin transporter (SLCO2A1) and hydroxyprostaglandin dehydrogenase-15 (HPGD). Endometrial biopsies were obtained from mares on day of ovulation (d0, n=4), late diestrus (LD, n=4), early luteolysis (EL, n=4) and after luteolysis (AL, n=4) during the cycle. Stages of the cycle were confirmed by plasma progesterone concentrations measured daily...
Lopes MA, Salter CE, Vandenplas ML, Berghaus R, Hurley DJ, Moore JN.To investigate whether expression of inflammation-associated genes in leukocytes from horses with gastrointestinal tract (GIT) diseases correlated with the type of disease and outcome. Methods: 10 healthy horses and 50 horses with GIT disease. Methods: A blood sample was collected from each healthy horse or horse with GIT disease (during admission to the hospital). Leukocytes were isolated, diluted to a standard concentration, and frozen until RNA extraction. Expression of 14 genes associated with inflammation was quantified by use of a real-time quantitative reverse transcription PCR assay. R...
Belacic ZA, Sullivan SN, Rice HC, Durgam SS.To investigate the effects of interleukin-1β (IL-1β) and methylprednisolone acetate (MPA) on equine intrabursal deep digital flexor tendon (DDFT) and navicular bone fibrocartilage (NBF) cells in vitro. Methods: Third passage DDFT and NBF cells from 5 healthy donor horses ages 11-17 years euthanized for reasons unrelated to musculoskeletal conditions. Methods: Aggregate cultures were incubated with culture medium alone (control), 10 ng/mL IL-1β, 10 ng/mL IL-1β + 0.05 mg/mL MPA, or 10 ng/mL IL-1β + 0.5 mg/mL MPA for 24 hours. Extracellular matrix (ECM) gene expressions were assessed via rea...
Gaede W, Reckling KF, Schliephake A, Missal D, Hotzel H, Sachse K.At a stud farm of Trakehner horses, all 33 foals of a birth cohort developed conjunctivitis and serous to muco-purulent rhinitis, and 7 older horses showed recurrent signs of conjunctivitis. Examination of nasal and conjunctival swabs by bacterial and cell culture, as well as real-time PCR, ArrayTube microarray analysis and DNA sequencing led to the identification of Chlamydophila (C.) caviae (first description in horses) and Streptococcus (S.) equi subsp. zooepidemicus. We presume a synergistic effect associated with these two agents by hypothesising that primary lesions were set by C. caviae...
Zhang H, Nagaoka K, Imakawa K, Nambo Y, Watanabe G, Taya K, Weng Q.The establishment of equine pregnancy is a unique and long process during which a series of physical and possibly biochemical interactions are required between the conceptus and uterus. In this study, we investigated the expression pattern of inhibin/activin subunits in the uterus during early pregnancy. The uteri from four adult mares on cyclic day 13 or pregnancy day 25 were obtained. Immunohistochemical experiments suggested that inhibin/activin subunits were immunolocalized in the luminal and glandular epithelium on pregnancy day 25. In addition, the inhibin α and inhibin/activin βB subu...
Austbø L, Røed KH, Dolvik NI, Skretting G.The aim of this study was to investigate genes for differential expression in cartilage of foals predisposed to osteochondrosis (OC). Tissue was sampled from the cranial part of the distal intermediate ridge of the tibia in the tarso-crural joint. Foals were considered predisposed to OC when parents had OC at the distal intermediate ridge of the tibia. RNA was isolated and subjected to arbitrarily primed PCR (RAP-PCR) followed by fingerprinting to screen for differentially expressed genes. By verification of results from the RAP-PCR fingerprint screening using real-time RT-PCR, we identified t...
Nadin-Davis S, Knowles MK, Burke T, Böse R, Devenish J.A quantitative real-time polymerase chain reaction method (qPCR) was developed and tested for the detection of Taylorella equigenitalis. It was shown to have an analytical sensitivity of 5 colony-forming units (CFU) of T. equigenitalis when applied to the testing of culture swabs that mimicked field samples, and a high analytical specificity in not reacting to 8 other commensal bacterial species associated with horses. As designed, it could also differentiate specifically between T. equigenitalis and T. asinigenitalis. The qPCR was compared to standard culture in a study that included 45 swab ...
Kolm G, Knapp E, Wagner R, Klein D.Lactoferrin (LF), a glycogen of the transferrin family with anti-bacterial and immunomodulatory properties, is expressed in various secretions and tissues. Cutaneous LF serves as a mast cell stabilising compound, modulates T cell activity and is found during IgE-mediated late phase reactions at allergen challenged sites. Culicoides hypersensitivity (CHS) in horses is a common IgE-mediated allergic dermatitis, characterised by an early and late phase cutaneous reaction upon allergen challenge. The aim of the study presented here was to examine whether LF mRNA expression in skin biopsies from ho...
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) ...
Silva CJD, Lima KPB, Monteiro JFDCLS, Silva AKSFD, Silva FJD, Pereira AMS, Hernandes VP, Silva EDD, Silva CSAGE, Brandão Filho SP, Brito MEF.American cutaneous leishmaniasis is a commonly neglected, vector-borne tropical parasitic disease that is a major public health concern in Brazil. Leishmania (Viannia) braziliensis is the main species associated with the disease. Accurate diagnosis is based on epidemiological surveillance, clinical assessment, and laboratory testing. Leishmania (V.) braziliensis has been detected in several wild and synanthropic mammals. Their epidemiological role has not been entirely elucidated. This study aimed to assess potential L. braziliensis infections in asymptomatic domestic animals, by molecular and...
Saulez MN, Godfroid J, Bosman A, Stiltner JL, Breathnach CC, Horohov DW.To determine concentrations of cytokine mRNA in horses with exercise-induced pulmonary hemorrhage (EIPH) after racing. Methods: 97 Thoroughbreds. Methods: Following tracheobronchoscopy, the severity of EIPH was graded (scale of 0 to 4), and venous blood samples were collected from 10 horses in each grade. After RNA isolation and cDNA synthesis, real-time PCR assay was conducted to detect cytokinespecific mRNA for interleukin (IL)-1, IL-6, and IL-10; interferon (INF)-gamma; and tumor necrosis factor (TNF)-alpha. Results: Neither location nor grade of EIPH affected the expression of IL-1 and INF...
Arai T, Hosoya M, Nakamura M, Magoori E, Uematsu Y, Sako T.The activities of the enzymes involved in the malate-aspartate shuttle and m RNA expression of malate dehydrogenase (MDH), a crucial enzyme for the NADH shuttle that produces ATP in glucose metabolism in the peripheral leukocytes of horses, were measured to investigate the change in metabolic states with training. There were no significant differences in plasma glucose and immunoreactive insulin concentrations between race horses and riding horses, used as a comparable reference. The cytosolic and mitochondrial MDH activities in leukocytes of race horses were significantly higher than those of...
Lopes MA, Salter CE, Vandenplas ML, Berghaus R, Hurley DJ, Moore JN.To investigate whether expression of inflammation-associated genes in leukocytes from horses with gastrointestinal tract (GIT) diseases correlated with the type of disease and outcome. Methods: 10 healthy horses and 50 horses with GIT disease. Methods: A blood sample was collected from each healthy horse or horse with GIT disease (during admission to the hospital). Leukocytes were isolated, diluted to a standard concentration, and frozen until RNA extraction. Expression of 14 genes associated with inflammation was quantified by use of a real-time quantitative reverse transcription PCR assay. R...
Mayo C, Venter E, Steyn J, Coetzee P, van Vuuren M, Crafford J, Schütte C, Venter G.The seasonal abundance of Culicoides midges, the vector of Bluetongue and African horse sickness viruses (BTV/AHSV) and the presence of viruses in midges were determined in 3 geographic areas in South Africa. In the Onderstepoort area, more than 500,000 Culicoides midges belonging to 27 species were collected. Eighteen midge species were collected throughout Winter and the presence of AHSV and BTV RNA in midges was detected using real time reverse transcription quantitative polymerase chain reaction. The nucleic acid of AHSV was found in 12 pools out of total pools of 35 Culicoides. Twenty‑f...
Aufox EE, Frank LA, May ER, Kania SA.Dermatophilus congolensis is a facultative anaerobic actinomycete that causes papular to exudative dermatitis with crusting in horses. This organism is frequently implicated as a cause of pastern dermatitis, but few data are available validating the organism's association with this disease. Objective: The aim of this study was to evaluate if D. congolensis is associated with pastern dermatitis in horses utilizing RT-qPCR. Methods: Fifteen client-owned horses diagnosed with pastern dermatitis and eight client-owned unaffected control horses were utilized for this study. Methods: A cross-sectio...
Ekiri AB, Long MT, Hernandez JA.The main objective of this study was to assess the diagnostic performance of a real-time polymerase chain reaction (PCR) assay for the detection of Salmonella in fecal samples collected from hospitalized horses with or without signs of gastrointestinal (GI) tract disease. The PCR assay used primers and a probe that targeted the invA gene of Salmonella. Assuming a sensitivity of 100% and a specificity of 96.6%, and a disease prevalence of 2%, 5%, and 10-15% in study horses, the PCR assay had a high (100%) negative predictive value, and a positive predictive value that ranged from 37% in horses ...
Riveroll AL, Skyba-Lewin S, Lynn KD, Mubyeyi G, Abd-El-Aziz A, Kibenge FST, Kibenge MJT, Cohen AM, Esparza-Gonsalez B, McD￮ L, Montelpare WJ.Adipose-derived stem cells (ADSCs) are used in tissue regeneration therapies. The objective of this study is to identify stable reference genes (RGs) for use in gene expression studies in a characterized equine adipose-derived mesenchymal stem cell (EADMSC) differentiation model. ADSCs were differentiated into adipocytes (ADs) or osteoblasts (OBs), and the proteomes from these cells were analyzed by liquid chromatography tandem mass spectrometry. Proteins that were stably expressed in all three cells types were identified, and the mRNA expression stabilities for their corresponding genes were ...
Knych HK, Harrison LM, Steinmetz SJ, Chouicha N, Kass PH.Clenbuterol, a beta2-adrenergic receptor agonist, is used therapeutically to treat respiratory conditions in the horse. However, by virtue of its mechanism of action it has been suggested that clenbuterol may also have repartitioning affects in horses and as such the potential to affect performance. Clenbuterol decreases the percent fat and increases fat-free mass following high dose administration in combination with intense exercise in horses. In the current study, microarray analysis and real-time PCR were used to study the temporal effects of low and high dose chronic clenbuterol administr...
Herrera-Luna CV, Budik S, Helmreich M, Walter I, Aurich C.Glucocorticoids (GCs) as mediators of the stress response may affect Leydig cell function by inhibiting either luteinizing hormone receptor expression or testosterone biosynthesis. The isozymes 11β-hydroxysteroid dehydrogenase (11βHSD) 1 and 11βHSD2 control the intracellular cortisol levels. Little is known about the effects of stress on fertility in the equine. The objective of the present study was to determine the presence and cellular localization of glucocorticoid receptors (GCR) and glucocorticoid-metabolizing enzymes (11βHSD1 and 11βHSD2) in equine epididymal and testicular tissue ...
Bone reportsJune 21, 2018
Volume 9 19-26 doi: 10.1016/j.bonr.2018.06.003
Semevolos SA, Duesterdieck-Zellmer KF, Larson M, Kinsley MA.Osteochondrosis (OC) is a naturally occurring disease of the articular-epiphyseal cartilage and subchondral bone layers, leading to pain and decreased mobility. The objective of this study was to characterize gene and protein expression of apoptotic markers in chondrocytes surrounding cartilage canals and along the osteochondral junction of osteochondrosis (OC)-affected and normal cartilage, using naturally occurring disease in horses. Paraffin-embedded osteochondral samples (6 OC, 8 normal controls) and cDNA from chondrocytes captured with laser capture microdissection (4 OC, 6 normal control...
Dzieciatkowski T, Przybylski M, Cymerys J, Turowska A, Chmielewska A, Tucholska A, Banbura MW.Equine herpesvirus type 1 (EHV-1) is one of the major viral agents causing diseases in horses common worldwide. A variety of techniques, including PCR, have been used to diagnose EHV-1 infections. In this paper, an attempt of real-time PCR has been described, which uses specific fluorochrome-labeled TaqMan probes for detection of viral DNA. This method does not require post-amplification manipulations, thereby reducing the risk of cross-contamination. The assay was sensitive enough to detect EHV-1 sequences in different clinical samples, as well in mice neuronal cell cultures. The technique wa...
Jose-Cunilleras E, Hayes KA, Toribio RE, Mathes LE, Hinchcliff KW.To clone and sequence cDNA for equine insulin-responsive glucose transporter (glucose transporter type 4 [GLUT-4]) and determine effects of glycogen-depleting exercise and meal type after exercise on GLUT-4 gene expression in skeletal muscle of horses. Methods: Muscle biopsy specimens from 7 healthy adult horses. Methods: Total RNA was extracted from specimens, and GLUT-4 cDNA was synthesized and sequenced. Horses were exercised on 3 consecutive days. On the third day of exercise, for 8 hours after exercise, horses were either not fed, fed half of daily energy requirements as hay, or fed an is...
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...
Alnaeem AA, Hemida MG.Equine infectious anemia virus (EIAV) is one of the most important threats to the equine industry globally. This is due to the poor performance of the affected horses, which requires euthanization of the infected animals upon the infection confirmation. Infected animals remain carriers throughout their life. EIAV infection has been reported in many parts of the world, including North America, Europe, Asia, and Africa. However, the EIAV status is never assessed in horses in the Gulf area, especially in the Kingdom of Saudi Arabia (KSA). Objective: This study aimed to perform molecular and serol...
Zhang L, Franchini M, Wehrli Eser M, Dip R.Accumulation of extracellular adenosine has been closely associated with human asthmatic responses. However, the relevance of adenosine signalling in equine airways has not previously been investigated. Objective: To determine the expression of adenosine receptors (AR) in bronchoalveolar lavage (BAL) cells and assess the reactivity of these cells to AR ligands ex vivo, employing IL-6 as readout of adenosinergic inflammatory signalling. Methods: Eight horses with varying degrees of lower airway inflammation and 10 healthy controls were analysed. Expression of AR-subtypes in each BAL sample was ...
Leon PM, Campos VF, Kaefer C, Begnini KR, McBride AJ, Dellagostin OA, Seixas FK, Deschamps JC, Collares T.The gene expression of Bax, Bcl-2, survivin and p53, following in vitro maturation of equine oocytes, was compared in morphologically distinct oocytes and cumulus cells. Cumulus-oocyte complexes (COC) were harvested and divided into two groups: G1 - morphologically healthy cells; and G2 - less viable cells or cells with some degree of atresia. Total RNA was isolated from both immature and in vitro matured COC and real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify gene expression. Our results showed there was significantly higher expression of survivin (P &...
Liu C, Cook FR, Cook SJ, Craigo JK, Even DL, Issel CJ, Montelaro RC, Horohov DW.Distinct from human lentivirus infection, equine infectious anemia virus (EIAV)-infected horses will eventually enter an inapparent carrier state in which virus replication is apparently controlled by adaptive immune responses. Although recrudescence of disease can occur after immune suppression, the actual immune correlate associated with protection has yet to be determined. Therefore, EIAV provides a model for investigating immune-mediated protective mechanisms against lentivirus infection. Here, we have developed a method to monitor EIAV-envelope specific cellular immunity in vivo. An EIA c...
Li S, Guo K, Wang X, Lin Y, Wang J, Wang Y, Du C, Hu Z, Wang X.Equine infectious anemia (EIA) has a worldwide distribution and causes severe economic losses to the equine industry. The EIA virus (EIAV) genome sequences from different countries are highly diverse, which poses a great challenge for pathogen identification with PCR. Phylogenetic analysis showed that although gag is the most conserved structural gene, it still has great genome variability. Currently, most existing PCR methods are designed based on the gag gene sequence and therefore do not cover all the viral strains, especially Asian EIAV strains. In this study, we developed a tat-gag-based ...
De Coster T, Van Poucke M, Bogado Pascottini O, Angel-Velez D, Van den Branden E, Peere S, Papas M, Gerits I, Govaere J, Peelman L, Vermeesch JR....In addition to fulfilling many breeders' curiosity, equine embryonic sex determination can have a profound commercial impact. However, the application of currently described assays for equine embryonic sexing has rendered variable diagnosis and validation rates, with sensitivity being the main problem. In addition, while pregnancy results of in vivo-flushed equine embryos following a needle aspiration biopsy equal those of non-biopsied embryos, the effect on in vitro-produced embryos is unknown. Here, we aimed to develop a highly sensitive and specific assay for equine sex determination that c...
is a tick-borne hemoparasite that causes equine piroplasmosis. It has a significant economic impact, decreasing performance and affecting animal welfare. This study aimed to identify DNA in the blood of horses from households in the southwestern and western regions of Romania. We included 310 animals, from which blood was collected via EDTA. To test the samples for the parasite genome, we used real-time PCR and conventional PCR. The prevalence of was 5.81% (18/310) in apparently healthy horses, suggesting that this parasite is enzootic in the regions studied, although veterinarians did not...
Salud publica de MexicoJune 26, 2007
Volume 49, Issue 3 210-217 doi: 10.1590/s0036-36342007000300006
Fernández-Salas I, de Lourdes Garza-Rodríguez M, Beaty BJ, Jiménez JR, Rivas-Estilla AM.To investigate the presence of WNV in birds, horses and humans in northeast Mexico. Methods: Serum samples from 33 birds, 24 horses and 237 humans were screened by ELISA for Anti-WNV antibodies. Human serum samples were also screened for WNV RNA using an RT-PCR assay. Results: Positive sera were found in three birds and 15 horses. Forty percent of the human serum samples were positive for IgG antibodies and 0% for IgM antibodies and viral RNA. Conclusions: The results of this study show that WNV is present in northeast Mexico and it is a new emergent infectious agent that represents a challeng...
