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 R, Quan M, Matjila PT, Zweygarth E, Guthrie AJ, Collins NE.Although a quantitative real-time PCR assay (qPCR) assay for the detection of Theileria equi has been developed and evaluated, it is possible that additional, as yet undetected 18S rRNA gene sequence variants may exist. A qPCR assay targeting a different gene, used in conjunction with the T. equi 18S rRNA qPCR assay, could assist in the detection of all T. equi genotypes in field samples. A T. equi ema-1-specific qPCR (Ueti et al., 2003) was tested on 107 South African field samples, 90 of which tested positive for T. equi antibody using the immuno-fluorescent antibody test (IFAT). The qPCR as...
Ousey JC, Palmer L, Cash RS, Grimes KJ, Fletcher AP, Barrelet A, Foote AK, Manning FM, Ricketts SW.Standard bacteriological methods for identifying Taylorella equigenitalis in cervical smears are time consuming. Therefore, a more rapid real-time PCR assay was evaluated for its suitability in screening swabs. Objective: To compare the results of a commercially available real-time PCR assay with routine microbiological culture for the identification of T. equigenitalis, the causative organism of contagious equine metritis, in equine genital swab samples, under 'field trial' conditions. Methods: Routine prebreeding genital swabs (n=2072) collected from Thoroughbred mares and stallions during 2...
Grønvold AM, L'Abée-Lund TM, Strand E, Sørum H, Yannarell AC, Mackie RI.Antimicrobial treatment is associated with the spread of antimicrobial resistance and disturbances in the ecological balance of intestinal microbiota. In horses, the main adverse effect of antimicrobial treatment is colitis. We used culture and 16S rRNA gene based molecular methods to monitor the prevalence of antimicrobial resistance and changes in predominant fecal populations during penicillin treatment and general anesthesia of horses in the clinical setting. After 5 days of parenteral administration of penicillin, fecal Escherichia coli were resistant to multiple unrelated antimicrobial a...
Echigoya Y, Okabe H, Itou T, Endo H, Sakai T.Muscle glycogen synthase (GYS1) is the rate-limiting enzyme in glycogen synthesis, and its activity is regulated by the phosphorylation states of certain amino acid residues encoded by the GYS1 gene. In the present study, the authors molecularly characterized the full-length equine GYS1 (eGYS1) cDNA and found that it contains a less common polyadenylation signal (AATACA). An amino acid alignment with other mammalian GYS1 showed that the phosphorylation sites in eGYS1 are completely conserved. Genomic DNA analysis revealed that the equine-specific substitutions (Glu 16 Asp and Ala 252 Thr) were...
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...
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...
Venugopal CS, Mendes LC, Peiró JR, Laborde SS, Stokes AM, Moore RM.To identify differentially expressed genes in pulmonary tissues of horses affected with summer pasture-associated obstructive pulmonary disease (SPAOPD), which is a form of recurrent airway obstruction (RAO), compared with those of unaffected horses. Methods: 6 horses with SPAOPD-RAO and 6 unaffected (healthy) horses. Methods: Horses were assigned to 2 groups on the basis of medical history, clinical score, and transpleural pressure. Total RNA from each of the 5 lung lobes of each of the 6 SPAOPD-RAO-affected horses was extracted and pooled. Similarly, total RNA from unaffected horses was pool...
Quan M, Lourens CW, MacLachlan NJ, Gardner IA, Guthrie AJ.Nucleotide sequences of 52 South African isolates of African horse sickness virus (AHSV) collected during 2004-2005 and including viruses of all nine AHSV serotypes, were used to design and develop a duplex real-time reverse transcription quantitative PCR (RT-PCR) assay targeting the VP7 (S8) and NS2 (S9) genes of AHSV. The assay was optimized for detection of AHSV in fresh and frozen blood of naturally infected horses. Assay performance was enhanced using random hexamers rather than gene-specific primers for RT, and with denaturation of double-stranded RNA in the presence of random hexamers. ...
Harris SP, Fujiwara N, Mealey RH, Alperin DC, Naka T, Goda R, Hines SA.Immune adult horses have CD8(+) cytotoxic T lymphocytes (CTLs) that recognize and lyse Rhodococcus equi-infected cells in an equine lymphocyte alloantigen (ELA)-A [classical major histocompatibility complex (MHC) class I]-unrestricted fashion. As protein antigens are MHC class I-restricted, the lack of restriction suggests that the bacterial antigens being recognized by the host are not proteins. The goals of this study were to test the hypothesis that these CTLs recognize unique R. equi cell-wall lipids related to mycobacterial lipids. Initial experiments showed that treatment of soluble R. e...
Quinlivan M, Maxwell G, Lyons P, Arkins S, Cullinane A.Equine rhinitis viruses (ERV) cause respiratory disease and loss of performance in horses. It has been suggested that the economic significance of these viruses may have been underestimated due to insensitive methods of detection. Objective: To develop a sensitive, rapid, real-time RT-PCR (rRT-PCR) assay suitable for the routine diagnosis and epidemiological surveillance of the A and B variants of ERV. Methods: TaqMan primer probe sets for ERAV and ERBV were designed from conserved regions of the 5' UTR of the ERV genome. Over 400 samples from both clinically affected and asymptomatic horses w...
Busschers E, Holt JP, Richardson DW.To determine effects of interleukin (IL)-1 beta and glucocorticoids on total glycosaminoglycan (GAG) loss and aggrecanase-mediated matrix degradation in equine cartilage. Methods: Cartilage from 24 equine cadavers free of sepsis and musculoskeletal disease. Methods: Effects of IL-1 beta, IL-1 beta with glucocorticoids (dexamethasone and triamcinolone, 10(-6) and 10(-7)M), and glucocorticoids alone on degradation of equine articular and nasal cartilage explants were assessed by measuring GAG release in media and GAG content in cartilage. Aggrecanase-mediated cleavage within the interglobular do...
Serteyn D, Piquemal D, Vanderheyden L, Lejeune JP, Verwilghen D, Sandersen C.Osteochondrosis (OC) is a developmental disease that affects growing horses and that severely affects their ability to perform. The genetic basis of its pathogenesis is poorly understood. The aim of the study was to analyze the transcript profile of leukocytes from horses affected with OC. Two transcriptome libraries were constructed from leukocytes of OC-affected and non-OC-affected horses using digital gene expression analysis (DGE) and real-time PCR. Statistical analysis allowed selection of 1,008 tags upregulated in the non-OC-affected group and 1,545 tags upregulated in the OC-affected gr...
Gerber V, De Feijter-Rupp H, Wagner J, Venta P, Harkema JR, Robinson NE.Airway mucus accumulation is associated with indoor irritant and allergen exposure in horses with recurrent airway obstruction (RAO). Epidermal growth factor receptor (EGFR) and a chloride channel (calcium activated, family member 1; CLCA1) are key signalling molecules involved in mucin gene expression. Objective: We hypothesised that exposure to irritants and aeroallergens would lead to increased expression of the mucin gene eqMUC5AC and increased stored mucosubstance in the airways of RAO-affected horses, associated with increased neutrophils and CLCA1 and EGFR mRNA levels. Methods: We perfo...
BMC research notesDecember 11, 2009
Volume 2 246 doi: 10.1186/1756-0500-2-246
Smits K, Goossens K, Van Soom A, Govaere J, Hoogewijs M, Vanhaesebrouck E, Galli C, Colleoni S, Vandesompele J, Peelman L.Application of reverse transcription quantitative real-time polymerase chain reaction is very well suited to reveal differences in gene expression between in vivo and in vitro produced embryos. Ultimately, this may lead to optimized equine assisted reproductive techniques. However, for a correct interpretation of the real-time PCR results, all data must be normalized, which is most reliably achieved by calculating the geometric mean of the most stable reference genes. In this study a set of reliable reference genes was identified for equine in vivo and fresh and frozen-thawed in vitro embryos....
Nieto JE, MacDonald MH, Braim AE, Aleman M.Horses are exquisitely sensitive to bacterial endotoxin and endotoxaemia is common in colic cases. In this study, gene expression of inflammatory cytokines was characterised in the blood of healthy horses following i.v. administration of lipopolysaccharide (LPS). Six horses received an LPS infusion and 6 controls received an equivalent volume of saline. Gene expression of genes encoding interleukin (IL)-1alpha, IL-1beta, IL-6, IL-8, and tumour necrosis factor-alpha (TNF-alpha) was quantified by real-time PCR. Gene expression of all inflammatory cytokines was upregulated following administratio...
Bhoora R, Quan M, Franssen L, Butler CM, van der Kolk JH, Guthrie AJ, Zweygarth E, Jongejan F, Collins NE.A quantitative real-time polymerase chain reaction (qPCR) assay using a TaqMan minor groove binder (MGB) probe was developed for the detection of Babesia caballi infection in equids from South Africa. Nine previously published sequences of the V4 hypervariable region of the B. caballi 18S rRNA gene were used to design primers and probes to target unique, conserved regions. The B. caballi TaqMan MGB qPCR assay was shown to be efficient and specific. The detection limit, defined as the concentration at which 95% of positive samples can be detected, was determined to be 0.000114% parasitized eryt...
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...
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...
Oliveira Filho JP, Badial PR, Cunha PHJ, Cruz TF, Araújo JP, Divers TJ, Winand NJ, Borges AS.Equine serum or plasma iron concentration drops quickly during inflammation. Accumulation of iron inside macrophages and reduction of the intestinal absorption of this element cause hypoferremia during systemic inflammatory processes. These mechanisms are mediated by hepcidin, a 25 amino acids peptide synthesized mainly in the liver in response to iron stores and inflammation. Hepcidin is an important peptide for systemic iron homeostasis and also has antibacterial and antifungal activities. Hepcidin up-regulation is particularly useful during acute inflammation, especially before adaptive imm...
Ramery E, Fievez L, Fraipont A, Bureau F, Lekeux P.The long pentraxin 3 (PTX3) plays an important role in host defence and its over-expression may contribute to airway injury. The aim of the present study was therefore to characterize in more detail PTX3 and its expression in the horses' airway. Six healthy horses and six horses affected by recurrent airway obstruction (R.A.O.) were submitted to a dusty environment challenge. PTX3 DNA and cDNA were cloned and sequenced. PTX3 expression was evaluated by RT-qPCR, Western blotting and immunohistochemistry in bronchoalveolar lavage fluid (BALF) cells, BALF supernatant and bronchial epithelial cell...
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...
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...
Lu Z, Chambers TM, Boliar S, Branscum AJ, Sturgill TL, Timoney PJ, Reedy SE, Tudor LR, Dubovi EJ, Vickers ML, Sells S, Balasuriya UB.The objective of this study was to develop and evaluate new TaqMan real-time reverse transcription-PCR (rRT-PCR) assays by the use of the minor groove binding probe to detect a wide range of equine influenza virus (EIV) strains comprising both subtypes of the virus (H3N8 and H7N7). A total of eight rRT-PCR assays were developed, targeting the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of the two EIV subtypes. None of the eight assays cross-reacted with any of the other known equine respiratory viruses. Three rRT-PCR assays (EqFlu NP, M, and HA3) which can detect strains of th...
Reyner CL, Wagner B, Young JC, Ainsworth DM.To examine gene expression of selected cytokines in pulmonary mononuclear cells isolated from healthy horses and horses susceptible to recurrent airway obstruction (RAO), and to determine whether interleukin (IL)-17 and IL-23 were associated with pulmonary inflammation. Methods: 6 RAO-susceptible and 5 healthy horses. Methods: Bronchoalveolar lavage cells were retrieved from horses that were stabled and fed dusty hay for 24 hours. Lavage cells devoid of neutrophils were incubated for 24 hours with solutions of PBS, hay dust, lipopolysaccharide, or B-glucan. Gene expression of IL-17, IL-23 (p19...
Pusterla N, Byrne BA, Hodzic E, Mapes S, Jang SS, Magdesian KG.A quantitative real-time (RT)-PCR assay was developed to detect Salmonella spp. in the feces of 911 equine species admitted to a veterinary hospital. Fresh feces and feces enriched for 24h in selenite broth were assessed by conventional culture and by RT-PCR targeting the Salmonella invA gene. The detection limit for the RT-PCR assay was 3 and 10 organisms, respectively, when spiked samples were purified from selenite broth and feces. The analytical specificity was 100% based on the detection of a panel of 40 salmonella serotypes from five serogroups and the lack of cross-reactivity with non-r...
Peretich AL, Abbott LL, Andrews FM, Dhar MS.To determine whether expression of mRNA for sodium-potassium adenosine-triphosphatase (NAKA) and sodium-hydrogen exchanger (NHE) in samples of the nonglandular portion of the equine gastric mucosa was altered by exposure to volatile fatty acids (VFAs) in an acidic environment. Methods: 10 horses (5 or = 12 years old). Methods: Samples of the nonglandular portion of the gastric mucosa were collected and exposed in Ussing chambers to Ringer's solution (control samples), Ringer's solution containing a mixture of VFAs (pH, 1.5 or 4.0), or Ringer's solution containing acetic acid (pH, 1.5 or 4.0)....
Pusterla N, Jackson R, Mapes SM, Noland J, Stenbom RM, Gebhart C.The humoral immune response and fecal shedding of Lawsonia intracellularis was investigated in 20 weanling foals following intra-rectal administration of frozen-thawed or lyophilized avirulent live L. intracellularis vaccine. Foals received either 30 mL frozen-thawed or lyophilized vaccine intra-rectally, given twice, 4 weeks apart. Serum samples from each foal were collected every 4 weeks for 16 weeks following the first vaccination and tested for anti-L. intracellularis specific IgG by immunoperoxidase monolayer assay. Rectal swabs were collected every other day following the first vaccinati...
Echigoya Y, Sato T, Itou T, Endo H, Sakai T.The species-specific properties of LDH isozymes are essentially determined by M (muscle) and H (heart) subunit proteins encoded by the LDHA and LDHB genes, respectively. In the present study, we molecularly characterized the full-length equine lactate dehydrogenase A (eLDHA) and B (eLDHB) cDNAs. The eLDHA cDNA consisted of a 999-bp open reading frame (ORF), while the eLDHB and newly acquired bat LDHB consisted of a 1002-bp ORF, which is 3 bp shorter than the LDHB ORF of other registered mammals. The alignment of amino acid sequences showed that eLDHA acquired positively charged His 88 and 226,...
van den Burg MM, Eizema K, de Graaf-Roelfsema E, van Breda E, Wijnberg ID, van der Kolk JH, Everts ME.To investigate the effects of acute exercise and long-term training on Na(+),K(+)-ATPase content, mRNA isoforms, and protein concentration in equine muscle. Methods: 6 Standardbreds. Methods: Horses performed a bout of exercise on a treadmill before and after 18 weeks of combined interval and endurance training. Muscle biopsy specimens were obtained from vastus lateralis muscle (VLM) and pectoralis descendens muscle (PDM) before and after exercise. The Na(+),K(+)-ATPase content, mRNA isoforms, and protein concentrations were determined by use of [(3)H]ouabain binding, real-time PCR assay, and ...
Diallo I, Read AJ, Kirkland PD.A real-time reverse transcription polymerase chain reaction (qRT-PCR) test for the matrix gene of type A influenza viruses was used during the 2007 Australian equine influenza (EI) outbreak in order to confirm diagnosis and, later, eradication of the virus. During the EI outbreak, horses being exported required vaccination and individual proof of freedom from EI. At the end of the outbreak, positive results were obtained from four horses destined for export, because of contamination of the samples with the vaccine. This report highlights the need for EI testing and vaccination to occur on sepa...
Klukowska-Rötzler J, Marti E, Lavoie JP, Ainsworth DM, Gerber V, Zurbriggen A, Janda J.Thymic stromal lymphopoietin (TSLP) is a cytokine involved in lymphocyte development. In humans and mice, TSLP drives the differentiation of T helper 2 (Th2) cells and the development of allergic inflammation. The equine TSLP gene has been previously identified and characterized, but its role in the pathogenesis of equine allergic diseases is not known. Our objective was to assess the expression of TSLP in bronchoalveolar lavage (BAL) cells and in primary bronchial epithelial cells (BEC) isolated from horses with recurrent airway obstruction (RAO). RNA was isolated from BAL cells sampled from ...
Brown LJ, Brown G, Kydd J, Stout TAE, Schulman ML.Equid herpesvirus type 1 is primarily a respiratory tract virus associated with poor athletic performance that can also cause late gestation abortion, neonatal foal death and encephalomyelopathy. Horizontal transmission is well described, whereas evidence of vertical transmission of equid herpesvirus type 1 associated with the birth of a healthy foal has not been demonstrated. This study sampled a population of Thoroughbred mares (n = 71), and their healthy neonatal foals and foetal membranes, to test for the presence of both equid herpesvirus types 1 and 4 using a quantitative polymerase chai...
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...
Tiwari M, Sodhi M, Verma P, Vivek P, Kataria RS, Niranjan SK, Bharti VK, Masharing N, Gujar G, Chanda D, Mukesh M.The identification of appropriate references genes is an integral component of any gene expression-based study for getting accuracy and reliability in data interpretation. In this study, we evaluated the expression stability of 10 candidate reference genes (GAPDH, RPL4, EEF1A1, RPS9, HPRT1, UXT, RPS23, B2M, RPS15, ACTB) in peripheral blood mononuclear cells of livestock species that are adapted to high altitude hypoxia conditions of Leh-Ladakh. A total of 37 PBMCs samples from six native livestock species of Leh-Ladakh region such as Ladakhi cattle, Ladakhi yak, Ladakhi donkey, Chanthangi goat...
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...
Sprekeler N, Müller T, Kowalewski MP, Liesegang A, Boos A.In many species, the small intestine is the major site of calcium (Ca(2+)) absorption. The horse differs considerably from most other species with regard to the physiology of its Ca(2+) metabolism and digestion. Thus, this study was performed to get more information about the transcellular Ca(2+) absorption in the horse.Two mechanisms of intestinal Ca(2+) absorption are described: the passive paracellular pathway and the active, vitamin D-dependent transcellular pathway. The latter involves the following elements: vitamin D receptors (VDR), transient receptor potential vanilloid channel member...
Grigolo B, Desando G, Cavallo C, Zini N, Ghisu S, Facchini A.Association of biomaterials with autologous cells can provide a new generation of implantable devices for cartilage repair. An ideal scaffold should possess a preformed three-dimensional shape, fix the cells to the damaged area and prevent their migration into the articular cavity. Furthermore, the constructs should have sufficient mechanical strength to facilitate handling in a clinical setting and stimulate the uniform spreading of cells and a phenotype re-differentiation process. The aim of this study was to verify the ability of an equine collagen membrane to support the growth of human ch...
Gerber V, De Feijter-Rupp H, Wagner J, Venta P, Harkema JR, Robinson NE.Airway mucus accumulation is associated with indoor irritant and allergen exposure in horses with recurrent airway obstruction (RAO). Epidermal growth factor receptor (EGFR) and a chloride channel (calcium activated, family member 1; CLCA1) are key signalling molecules involved in mucin gene expression. Objective: We hypothesised that exposure to irritants and aeroallergens would lead to increased expression of the mucin gene eqMUC5AC and increased stored mucosubstance in the airways of RAO-affected horses, associated with increased neutrophils and CLCA1 and EGFR mRNA levels. Methods: We perfo...
Campolo A, de Laat MA, Keith L, Gruntmeir KJ, Lacombe VA.Insulin dysregulation is common in horses although the mechanisms of metabolic dysfunction are poorly understood. We hypothesized that insulin signaling in striated (cardiac and skeletal) muscle and lamellae may be mediated through different receptors as a result of receptor content, and that transcriptional regulation of downstream signal transduction and glucose transport may also differ between tissues sites during hyperinsulinemia. Archived samples from horses treated with a prolonged insulin infusion or a balanced electrolyte solution were used. All treated horses developed marked hyperin...
Tewari D, Del Piero F, Cieply S, Feria W, Acland H.Equine herpesvirus-1 (EHV-1) strains with a single point mutation at the 2254 nucleotide position with a G2254 constitution within the DNA polymerase gene are associated strongly with equine myeloencephalopathies. Infections with non-neuropathogenic EHV-1 strains without the G2254 nucleotide but with an A2254 nucleotide are associated less frequently with equine neurologic disease. A retrospective study utilizing DNA extracted from formalin fixed paraffin embedded tissues was conducted with real time PCR and pyrosequencing, to determine the infecting EHV-1 strains. Infection with EHV-1 A2254 a...
Alnaeem A, Shawaf T, Ali AM, Hemida MG.In the current study, we are investigating the viral causes of some respiratory clinical signs in some animals belongs to the family Equidae in eastern Saudi Arabia (ESA) during winter- 2019. We observed the progression of severe respiratory clinical signs among some horses, donkeys, and ponies in the ESA. Animals showed rapid respiration, fever, nasal discharges (started as serous then changed into mucopurulent with the progression of the infection per some animals). We conducted a longitudinal study to monitor the progression of this outbreak. We conducted molecular surveillance for the infl...
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...
Migdał A, Migdał Ł, Oczkowicz M, Okólski A, Chełmońska-Soyta A.The aim of this study was to investigate the molecular mechanisms leading to the identification of pathogens by congenital immune receptors in foals up to 60 days of age. The study was conducted on 16 foal Polish Pony Horses (Polish Konik) divided into two study groups: control ( = 9) and experimental ( = 7). Foals from the experimental group received an intramuscular duplicate injection of 5 mL of Biotropina (Biowet) at 35 and 40 days of age. The RNA isolated from venous blood was used to evaluate the expression of the, , and genes using RT-PCR. The results of the experiment demonstrated a s...
Herbst AC, Reedy SE, Page AE, Horohov DW, Adams AA.Studies investigating age-related changes in the function of monocytes are currently limited for horses. Thus, the main goal of this study was to determine the effect of aging on monocyte phagocytic capacity and pro-inflammatory cytokine responses to bacterial lipopolysaccharide (LPS). A second goal of this work was to examine the effect of aging on the inflammatory cytokine responses to LPS in a whole blood ex vivo model. Seven healthy young adult (4-6 years of age) and seven healthy senior horses (>20 years of age) were enrolled. Phagocytosis of E. coli, and pro-inflammatory cytokine (TNF...
Amaral A, Fernandes C, Szóstek-Mioduchowska A, Lukasik K, Rebordão MR, Pinto-Bravo P, Skarzynski DJ, Ferreira-Dias G.Cathepsin G (CAT) is a protease released by neutrophils when forming neutrophil extracellular traps that was already associated with inducing type I collagen (COL1) in equine endometrium in vitro. Endometrosis is a fibrotic condition mainly characterized by COL1 deposition in the equine endometrium. The objective was to evaluate if noscapine (an alkaloid for cough treatment with anti-neoplastic and anti-fibrotic properties) would reduce transcription (evaluated by qPCR) and COL1 protein relative abundance (evaluated by western blot) induced by CAT in equine endometrial explants from follicula...
Tachibana Y, Sakurai T, Bai H, Shiota K, Nambo Y, Nagaoka K, Imakawa K.Extensive studies have been conducted to characterize the unique phenomena of equine pregnancy. Most studies have focused on embryo transmigration when the embryo is covered with a mucin-like glycoprotein capsule and on the characterization of the chorionic girdle and chorionic gonadotropin (CG) secretion. However, the events preceding and following capsule disappearance have not been well studied. In this study, the mRNA expression in conceptus membranes at days 19, 21, and 25 (day 0 = day of ovulation) was analyzed by RNA-seq (SOLiD3), and transcript levels on these three days and day 13...
Trachsel DS, Stage HJ, Rausch S, Trappe S, Söllig K, Sponder G, Merle R, Aschenbach JR, Gehlen H.Physiological particularities of the equine heart justify the development of an in vitro model suitable for investigations of the species-specific equine cardiac electrophysiology. Adipose tissue-derived stromal/stem cells (ASCs) could be a promising starting point from which to develop such a cardiomyocyte (CM)-like cell model. Therefore, we compared abdominal, retrobulbar, and subcutaneous adipose tissue as sources for the isolation of ASCs applying two isolation methods: the collagenase digestion and direct explant culture. Abdominal adipose tissue was most suitable for the isolation of ASC...
Ellero N, Lanci A, Baldassarro VA, Alastra G, Mariella J, Cescatti M, Giardino L, Castagnetti C.The importance of trophic factors, such as nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and brain-derived neurotrophic factor (BDNF) during the perinatal period, is now emerging. Through their functional activities of neurogenesis and angiogenesis, they play a key role in the final maturation of the nervous and vascular systems. The present study aims to: (i) evaluate the NGF and VEGF levels obtained at parturition from the mare, foal and umbilical cord vein plasma, as well as in amniotic fluid; (ii) evaluate NGF and VEGF content in the plasma of healthy foals during t...
Fukuda K, Mita H, Tamura N, Kuroda T, Kuwano A, Takahashi T, Sato F.Persistent synovitis damages the articular cartilage in horses. To evaluate the effectiveness of treatment for synovitis using a model induced by intra-articular administration of monoiodoacetic acid (MIA), it is necessary to identify inflammatory biomarkers characteristic of the MIA model. Synovitis was induced by administering MIA into the unilateral antebrachiocarpal joints of five horses, and saline was injected into the contralateral joints as a control on day 0. Clinical and ultrasonographic examinations and synovial fluid collection were performed on days 0, 1, 2, 7, 14, 21, 28, and 35....
Kim SH, Lee JS, Lee JH, Kim YJ, Choi JG, Lee SK, Kim HJ, Yang SJ, Park T, Lee SK, Kang HE, Jeoung HY, Park JY.Venereal diseases caused by bacteria are important to the equine industry due to economic losses caused by decline of conception rate in breeding horses. Therefore, identification of infected animals as well as the implementation of appropriate managerial procedures based on accurate diagnosis is critical. In this study, two types of multiplex real-time polymerase chain reaction with high sensitivity and specificity were developed for the simultaneous detection and differentiation of five commonly associated bacterial pathogens of venereal diseases in horses, consisting of Taylorella equigenit...
Beg MA, Gastal EL, Gastal MO, Ji S, Wiltbank MC, Ginther OJ.Transvaginal ultrasound-guided luteal biopsy was used to evaluate the effects of prostaglandin (PG)F2alpha on steady-state concentrations of mRNA for specific genes that may be involved in regression of the corpus luteum (CL). Eight days after ovulation (Hour 0), mares (n=8/group) were randomized into three groups: control (no treatment or biopsy), saline+biopsy (saline treatment at Hour 0 and luteal biopsy at Hour 12), or PGF2alpha+biopsy (5mg PGF2alpha at Hour 0 and luteal biopsy at Hour 12). The effects of biopsy on CL were compared between the controls (no biopsy) and saline+biopsy group. ...
Leisinger CA, Klein C, Markle ML, Premanandan C, Sones JL, Pinto CRF, Paccamonti DL.Aluteal cycles were induced in the mare to evaluate the effects of progesterone deprivation on the gene expression of embryos and endometrium collected eight days after ovulation. We hypothesized that the transcript expression would be altered during induced aluteal (AL) cycles (low progesterone <1 ng/mL) when compared with control cycles during diestrus (high progesterone; > 4 ng/mL) for 1) the embryonic expression of progesterone-mediated transcripts and those related to normal embryo growth and development and 2) the endometrial expression of progesterone-mediated transcripts and those ...
Ramery E, Fievez L, Fraipont A, Bureau F, Lekeux P.The long pentraxin 3 (PTX3) plays an important role in host defence and its over-expression may contribute to airway injury. The aim of the present study was therefore to characterize in more detail PTX3 and its expression in the horses' airway. Six healthy horses and six horses affected by recurrent airway obstruction (R.A.O.) were submitted to a dusty environment challenge. PTX3 DNA and cDNA were cloned and sequenced. PTX3 expression was evaluated by RT-qPCR, Western blotting and immunohistochemistry in bronchoalveolar lavage fluid (BALF) cells, BALF supernatant and bronchial epithelial cell...
Williams JM, Lin YJ, Loftus JP, Faleiros RR, Peroni JF, Hubbell JA, Ravis WR, Belknap JK.Laminitis is a serious complication of horses suffering from sepsis/endotoxaemia-related events. Laminitis in horses and organ injury in human sepsis are both reported to involve inflammatory injury to the laminae/organs including early activation of endothelium and leucocytes leading to emigration of neutrophils into the tissue interstitium. In the black walnut extract (BWE) model, systemic inflammatory events coincide with marked increase in laminar mRNA concentrations of inflammatory genes including proinflammatory cytokines (i.e. IL-1beta, IL-6), COX-2, chemokines (i.e. IL-8) and endotheli...
Diel de Amorim M, Bramer SA, Rajamanickam GD, Klein C, Card C.Our objectives were to examine changes in endometrial and luteal gene expression during estrus, diestrus, pregnancy and treatments to induce luteolysis and putatively induce luteostasis. Groups were: Diestrus (DIEST), Estrus (ESTR), Pregnant (PREG), Oxytocin (OXY), Carbetocin (CARB), and Meclofenamic acid (MFA). Blood was obtained from day (D)12 to D15 for measurement of oxytocinase, also referred to as leucyl-cysteinyl aminopeptidase (LNPEP) and progesterone. Luteal biopsies were obtained on D12 and D15 and an endometrial biopsy on D15. Real-time RT-PCR was performed for the following genes: ...
Echigoya Y, Okabe H, Itou T, Endo H, Sakai T.Muscle glycogen synthase (GYS1) is the rate-limiting enzyme in glycogen synthesis, and its activity is regulated by the phosphorylation states of certain amino acid residues encoded by the GYS1 gene. In the present study, the authors molecularly characterized the full-length equine GYS1 (eGYS1) cDNA and found that it contains a less common polyadenylation signal (AATACA). An amino acid alignment with other mammalian GYS1 showed that the phosphorylation sites in eGYS1 are completely conserved. Genomic DNA analysis revealed that the equine-specific substitutions (Glu 16 Asp and Ala 252 Thr) were...
Kennedy R, Lappin DF, Dixon PM, Bennett D, Riggio MP.Equine periodontitis is a common and painful condition. However, the disease often goes unnoticed by owners and is thus a major welfare concern. The aetiopathogenesis of the condition remains poorly understood and has been investigated in few studies. The innate immune system is known to play an important role in human periodontitis, but its role in equine periodontitis has not been examined. Objective: To quantify the messenger (m)RNA levels of Toll-like receptors (TLRs) and cytokines in gingival tissue from orally healthy horses and those affected by periodontitis. Methods: Observational stu...
ERas is a new gene of the Ras family found in murine embryonic stem (ES) cells. Its human ortholog is not expressed in human ES cells. So far ERas gene has only been found to be expressed in the tissues of adult cynomolgus monkeys and cattle; however, information about ERAS expression or its potential functions in equine tissues is lacking. This study was performed to investigate whether Eras is an equine functional gene and whether ERAS is expressed in the tissues of adult horses and determine its potential physiological role. Expression of the ERas gene was detected in all examined adult tis...
Morris ERA, Schroeder ME, Ferro PJ, Waller AS, McGlennon AA, Bustos CP, Gressler LT, Wu J, Lawhon SD, Boyle AG, Lingsweiler S, Paul N, Dimitrov K....Strangles is a contagious bacterial disease of horses caused by Streptococcus equi subspecies equi (SEE) that occurs globally. Rapid and accurate identification of infected horses is essential for controlling strangles. Because of limitations of existing PCR assays for SEE, we sought to identify novel primers and probes that enable simultaneous detection and differentiation of infection with SEE and S. equi subsp. zooepidemicus (SEZ). Comparative genomics of U.S. strains of SEE and SEZ (n = 50 each) identified SE00768 from SEE and comB from SEZ as target genes. Primers and probes for real-ti...
