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.
Hue ES, Fortier CI, Laurent AM, Quesnelle YF, Fortier GD, Legrand LJ, Pronost SL.The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 norm. After the development and first validation step, two distinct characterization steps were performed according to the AFNOR norm: (a) characterization of the qRT-PCR assay alone and (b) characterization of the whole analytical method. The validation of the whole analytical method included the portrayal of all steps between the extraction of nucleic acids and the final PCR analysis. Validation of the whole method is very important for vi...
Spaas JH, Gomiero C, Broeckx SY, Van Hecke L, Maccatrozzo L, Martens A, Martinello T, Patruno M.Several cytokines and growth factors play an essential role in skin regeneration and epithelial-like stem cells (EpSCs) have beneficial effects on wound healing in horses. However, there are no reports available on the expression of these growth factors and cytokines after EpSC therapy. Methods: Wounds of 6 cm(2) were induced in the gluteus region of 6 horses and treated with (i) autologous EpSCs, (ii) allogeneic EpSCs, (iii) vehicle treatment or (iv) untreated control. Real time polymerase chain reaction was performed on tissue biopsies taken 1 and 5 weeks after these treatments to evaluate...
Okabe K, Mukai K, Ohmura H, Takahashi T, Miyata H.We examined the effect of transient hypoxic exposure during high-intensity exercise on satellite cell activation and angiogenesis in Thoroughbred skeletal muscle. Methods: Six Thoroughbreds horses (6.3±0.8 years old) ran on a treadmill in normoxia (N; FIO2=21%) and hypoxia (H; FIO2=16%) at the same speed for the same duration, and oxygen consumption and plasma lactate and hemoglobin concentrations were measured. In addition, muscle biopsy samples were obtained from gluteus medius muscle before exercise and immediately, 4 hours, 24 hours, 3 days and 7 days after exercise, and immunohistochemic...
Stasiak K, Rola J, Zmudzinski JF.A highly sensitive and specific real-time PCR assay was used for detection and quantitation of equine herpesvirus type 1 (EHV-1) in the different internal organs of aborted fetuses. Tissue samples from 23 aborted fetuses submitted to the Department of Virology of the National Veterinary Research Institute in Pulawy between 2012 and 2013 were used for testing. Total DNA was extracted using a phenol-chloroform-isoamyl alcohol standard protocol. A real-time PCR with forward and reverse primers encompassing a highly conserved region encoding viral glycoprotein B was adapted for diagnosis of EHV-1 ...
Madrigal RG, Shaw SD, Witkowski LA, Sisson BE, Blodgett GP, Chaffin MK, Cohen ND.Current screening tests for Rhodococcus equi pneumonia in foals lack adequate accuracy for clinical use. Real-time, quantitative PCR (qPCR) for virulent R. equi in feces has not been systematically evaluated as a screening test. Objective: The objective of this study was to evaluate the accuracy of qPCR for vapA in serially collected fecal samples as a screening test for R. equi pneumonia in foals. Methods: One hundred and twenty-five foals born in 2011 at a ranch in Texas. Methods: Fecal samples were collected concurrently with thoracic ultrasonography (TUS) screening examinations at ages 3, ...
Babesia caballi and Theileria equi are tick-borne pathogens, etiological agents of equine piroplasmosis that affect different species of Equidae causing relevantly important direct and indirect losses. A field study was conducted to evaluate the distribution of the equine piroplasms in an area of Central-Southern Italy and to identify correlated risk factors. Serum samples of 673 asymptomatic horses were collected during spring-summer of 2013 to estimate the seroprevalence of the parasites within the study area using T. equi and B. caballi Antibody test kit (VMRD(®), Inc, Pullman, WA, USA). T...
Aeschbacher S, Santschi E, Gerber V, Stalder HP, Zanoni RG.Equine influenza is a highly contagious respiratory disease in horses caused by influenza A viruses. In this work a real-time RT-PCR for fast and sensitive diagnosis of equine influenza viruses (EIV) targeting a highly conserved region of the matrix gene was developed. In addition two RT-PCR methods for the amplification of large parts of the matrix- and HA gene were adapted for molecular-epidemiological characterization of viruses. The primers of the real-time RT-PCR had homologies of 99.4% to EIV- and 97.7% to all influenza A viral sequences, whereas the minor groove binder (MGB) probe showe...
Stefańska I, Witkowski L, Rzewuska M, Dzieciątkowski T.Rhodococcus equi is the causative agent of rhodococcosis in horses, resulting in significant morbidity and mortality in foals. This bacterium has also been isolated from a variety of animals and is being increasingly reported as a cause of infection in humans, mainly in immunosuppressed individuals. Laboratory diagnostics of R. equi infections based only on conventional microbiological methods shows low accuracy and can lead to misidentification. The objective of the study was to develop and evaluate a real-time PCR assay for direct detection of R. equi in various clinical specimens, including...
Barton MH, Darden JE, Clifton S, Vandenplas M.To validate primer sets for use in reverse transcription quantitative PCR assays to measure gene expression of cytosolic phospholipase A2 (cPLA2) and microsomal prostaglandin E2 synthase 1 (mPGES1) in equine mononuclear cells and determine the effects of firocoxib, a selective cyclooxygenase 2 (COX-2) inhibitor, on COX-2, cPLA2, and mPGES1 gene expression following incubation of mononuclear cells with lipopolysaccharide (LPS). Methods: 8 healthy adult horses. Methods: Peripheral blood mononuclear cells were isolated by density gradient centrifugation and incubated at 37°C with medium alone, f...
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 ...
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...
Balasuriya UB, Crossley BM, Timoney PJ.Equid herpesvirus 1 (EHV-1) is one of the most economically important equine viral pathogens. Its clinical manifestations in horses vary from acute upper respiratory tract disease, abortion, or neonatal death, to neurological disease termed equine herpesviral myeloencephalopathy, which may lead to paralysis and a fatal outcome. Successful identification of EHV-1 infection in horses depends on a variety of factors such as suitable case selection with emphasis on timing of sample collection, selection of appropriate sample(s) based on the clinical manifestations, application of relevant diagnost...
Shaw SD, Cohen ND, Chaffin MK, Blodgett GP, Syndergaard M, Hurych D.Real-time, quantitative PCR (qPCR) methods for detecting Rhodococcus equi in feces have been developed as a noninvasive, rapid diagnostic test for R. equi pneumonia, but have not been evaluated in a large population of foals. Objective: The objective of this study was to evaluate the clinical utility of fecal PCR as a diagnostic test for R. equi pneumonia in foals using receiver operating characteristic (ROC) methods. Methods: 186 foals born in 2011 at an R. equi-endemic ranch in Texas. Methods: Fecal samples were collected at the time of onset of clinical signs for pneumonic foals (n = 3...
Cieslak J, Mackowski M, Czyzak-Runowska G, Wojtowski J, Puppel K, Kuczynska B, Pawlak P.Apart from the well-known role of somatic cell count as a parameter reflecting the inflammatory status of the mammary gland, the composition of cells isolated from milk is considered as a valuable material for gene expression studies in mammals. Due to its unique composition, in recent years an increasing interest in mare's milk consumption has been observed. Thus, investigating the genetic background of horse's milk variability presents and interesting study model. Relying on 39 milk samples collected from mares representing three breeds (Polish Primitive Horse, Polish Cold-blooded Horse, Pol...
Vieira RF, Vidotto O, Vieira TS, Guimaraes AM, Santos AP, Nascimento NC, Santos NJ, Martins TF, Labruna MB, Marcondes M, Biondo AW, Messick JB.The aims of this study were to determine the prevalence of hemoplasmas in a rural Brazilian settlement's population of human beings, their dogs and horses, highly exposed to tick bites; to identify the tick species parasitizing dogs and horses, and analyze factors associated with their infection. Blood samples from 132 dogs, 16 horses and 100 humans were screened using a pan-hemoplasma SYBR green real-time PCR assay followed by a species-specific TaqMan real-time PCR. A total of 59/132 (44.7%) dog samples were positive for hemoplasmas (21 Mycoplasma haemocanis alone, 12 ' Candidatus Mycoplasma...
Li J, Kelly P, Zhang J, Xu C, Wang C.Babesia spp. are tick-borne protozoan hemoparasites and the second most common blood-borne parasites of mammals, in particular domestic animals. We used the Clustal Multiple Alignment program and 18S rRNA gene sequences of 22 Babesia species from GenBank to develop a PCR that could detect a wide variety of Babesia spp. in a single reaction. The pan-Babesia FRET-qPCR we developed reliably detected B. gibsoni, B. canis, B. vogeli, B. microti, B. bovis, and B. divergens under controlled conditions but did not react with closely related species, mainly Hepatozoon americanum, Theileria equi, and To...
Perglione CO, Gildea S, Rimondi A, Miño S, Vissani A, Carossino M, Cullinane A, Barrandeguy M.In 2012, equine influenza (EI) virus was confirmed as the cause of outbreaks of respiratory disease in horses throughout South America. In Uruguay and Argentina, hundreds of vaccinated thoroughbred horses in training and racing facilities were clinically affected. Objective: To characterise the EI viruses detected during the outbreak in Uruguay and Argentina. Methods: Virus was detected in nasopharyngeal swabs by a pan-reactive influenza type A real-time RT-PCR. The nucleotide sequence of the HA1 gene was determined and analysed phylogenetically using mega 5 software. Amino acid sequences alig...
Maia VN, Batista AM, Cunha Neto S, Silva DM, Adrião M, Wischral A.The effects of deslorelin acetate use in inducing ovulation need to be clarified to improve the results of equine embryo transfer. The mRNA abundance for angiogenic factors and LH receptor (LHR) in corpus luteum (CL) was studied in mares with natural (control group [CG]) and induced ovulation with deslorelin acetate (treatment group [TG]; follicles: ≥ 35 mm). Transrectal ultrasonography was used to verify the ovulation day, and on Days 4, 8, and 12 after ovulation (Day 0), CL samples were obtained through ultrasound-guided biopsy. The messenger RNA expression of vascular endothelial growth f...
Rushton JO, Kolodziejek J, Nell B, Weissenböck H, Nowotny N.The role of equid γ-herpesviruses on ocular surface diseases has been disputed, because the diagnosis is usually based on clinical symptoms and detection of viral DNA from samples obtained from live animals. Objective: To describe the clinical course, results of polymerase chain reaction (PCR) analysis, in situ hybridisation, cell culture and pathohistological findings of select cases in a presumed outbreak of herpesvirus infection in a group of 15 Icelandic horses. Methods: Case series. Methods: Pooled ocular and nasal swabs and peripheral blood mononuclear cells of horses diagnosed clinica...
Kullmann A, Weber PS, Bishop JB, Roux TM, Norby B, Burns TA, McCutcheon LJ, Belknap JK, Geor RJ.Hyperinsulinaemia is implicated in the pathogenesis of endocrinopathic laminitis. Insulin can bind to different receptors: two insulin receptor isoforms (InsR-A and InsR-B), insulin-like growth factor-1 receptor (IGF-1R) and InsR/IGF-1R hybrid receptor (Hybrid). Currently, mRNA expression of these receptors in equine tissues and the influence of body type and dietary carbohydrate intake on expression of these receptors is not known. Objective: The study objectives were to characterise InsR-A, InsR-B, IGF-1R and Hybrid expression in lamellar tissue (LT) and insulin responsive tissues from horse...
Weyer CT, Joone C, Lourens CW, Monyai MS, Koekemoer O, Grewar JD, van Schalkwyk A, Majiwa PO, MacLachlan NJ, Guthrie AJ.Blood samples collected as part of routine diagnostic investigations from South African horses with clinical signs suggestive of African horse sickness (AHS) were subjected to analysis with an AHS virus (AHSV) group specific reverse transcription quantitative polymerase chain reaction (AHSV RT-qPCR) assay and virus isolation (VI) with subsequent serotyping by plaque inhibition (PI) assays using AHSV serotype-specific antisera. Blood samples that tested positive by AHSV RT-qPCR were then selected for analysis using AHSV type specific RT-qPCR (AHSV TS RT-qPCR) assays. The TS RT-qPCR assays were ...
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...
Herrera-Luna CV, Scarlet D, Walter I, Aurich C.The aim of the present study was to characterise receptors for LH and FSH (LHR and FSHR, respectively) and aromatase in epididymal and testicular tissue from stallions of different ages (prepubertal, young, mature and old). Gene and protein expression were assessed by real-time quantitative polymerase chain reaction (real-time qPCR), immunohistochemistry and multiple immunofluorescence labelling. There were no differences in LHR mRNA expression in epididymal and testicular parenchyma in stallions of different age. In contrast, expression of FSHR and CYP19A1 in caput, corpus and cauda epididymi...
Olofsson KM, Hjertner B, Fossum C, Press CM, Lindberg R.Inflammatory bowel disease (IBD) in horses is an idiopathic disorder, encompassing different types of chronic intestinal inflammation. The pathogenesis of the disease remains to be established, but it has been suggested that an imbalance between regulatory T cells (Tregs) and T helper 17 (Th17)-associated cytokines and altered toll-like receptor 4 (TLR4) expression is associated with intestinal inflammation in other species. The aim of the present study was to quantify Tregs in rectal biopsies from horses affected with IBD by immunohistochemistry and to evaluate expression of genes encoding in...
Stephenson N, Hodzic E, Mapes S, Rejmanek D, Foley J.Anaplasma phagocytophilum is a tick-transmitted bacterial pathogen of humans and animals comprising strains that cause clinical disease in people, dogs and horses (the pathogenic A. phagocytophilum "genospecies") and more distantly related strains. A rodent-adapted genospecies named DU1, found primarily in woodrats, is unable to infect horses. We developed a real-time PCR (RT-PCR) assay, which targets an 85 base pair region of the ank gene and is specific for the pathogenic genospecies of A. phagocytophilum from North America. Thirty DNA samples from A. phagocytophilum RT-PCR-positive rodents ...
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 ...
Andreassen SM, Berg LC, Nielsen SS, Kristensen AT, Jacobsen S.Studies in humans have shown that haemostatic and inflammatory pathways both play important roles in the pathogenesis of joint disease. The aim of this study was to assess mRNA expression of haemostatic and inflammatory factors in cultured equine fibroblast-like synoviocytes exposed to lipopolysaccharide (LPS), fibrinogen and thrombin. Synovial membranes were collected from metacarpo-phalangeal joints of 6 skeletally mature horses euthanized for non-orthopaedic reasons. Passage 4 fibroblast-like synoviocytes were left non-treated or treated with either 0.1 μg/ml LPS, 5 mg/ml fibrinogen or 5 U...
Nazari F, Parham A, Maleki AF.Quantitative real time reverse transcription PCR (qRT-PCR) is one of the most important techniques for gene-expression analysis in molecular based studies. Selecting a proper internal control gene for normalizing data is a crucial step in gene expression analysis via this method. The expression levels of reference genes should be remained constant among cells in different tissues. However, it seems that the location of cells in different tissues might influence their expression. The purpose of this study was to determine whether the source of mesenchymal stem cells (MSCs) has any effect on exp...
Drebert Z, Golke A, Cymerys J, Słońska A, Chmielewska A, Tucholska A, Bańbura MW.Equid herpesvirus type 1 (EHV-1) is a prevalent causative agent of equine diseases worldwide. After primary replication in the respiratory epithelium the virus disseminates systemically through a peripheral blood mononuclear cell (PBMC)-associated viraemia. EHV-1 is the only alphaherpes- virus known so far which is capable of establishing latent infection not only in neurons but also in immune system cells (mainly in lymphocytes and macrophages). Since leukocytes are not the target cells for viral replication but are used to transport EHV-1 to the internal organs, the questionremains how the v...
Remacha AR, Barrachina L, Álvarez-Arguedas S, Ranera B, Romero A, Vázquez FJ, Zaragoza P, Yañez R, Martín-Burriel I, Rodellar C.The immunomodulatory capacities of mesenchymal stem cells (MSCs) have made them the subject of increased clinical interest for tissue regeneration and repair. We have studied the immunomodulatory capacity of equine MSCs derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) in cocultures with allogeneic peripheral blood mononuclear cells (PBMCs). Different isoforms and concentrations of phytohaemaglutinin (PHA) were tested to determine the best stimulation conditions for PBMC proliferation and a proliferation assay was performed for 7 days to determine the optimal day of stimulation o...
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...
Tozaki T, Ohnuma A, Iwai S, Kikuchi M, Ishige T, Kakoi H, Hirota K, Kusano K, Nagata S.Gene doping is banned in human sports, horseracing, and equestrian sports. One possible form of gene doping is to administer exogenous genes, called transgenes. Several transgene detection methods based on quantitative PCR have been developed. In this study, we investigated the robustness of digital PCR and real-time PCR in transgene detection using primers and probes that matched (P-true) or incompletely matched (P-false) the template DNA. Fluorescence intensity was significantly reduced when substituted probes were used compared to that using the matched probe in both digital and real-time P...
Noronha LE, Harman RM, Wagner B, Antczak DF.The immunoreceptor NKp46 is considered to be the most consistent marker of NK cells across mammalian species. Here, we use a recombinant NKp46 protein to generate a panel of monoclonal antibodies that recognize equine NKp46. The extracellular region of equine NKp46 was expressed with equine IL-4 as a recombinant fusion protein (rIL-4/NKp46) and used as an immunogen to generate mouse monoclonal antibodies (mAbs). MAbs were first screened by ELISA for an ability to recognize NKp46, but not IL-4, or the structurally related immunoreceptor CD16. Nine mAbs were selected and were shown to recognize ...
Mayo CE, Weyer CT, Carpenter MJ, Reed KJ, Rodgers CP, Lovett KM, Guthrie AJ, Mullens BA, Barker CM, Reisen WK, MacLachlan NJ.The availability of rapid, highly sensitive and specific molecular and serologic diagnostic assays, such as competitive enzyme-linked immunosorbent assay (cELISA), has expedited the diagnosis of emerging transboundary animal diseases, including bluetongue (BT) and African horse sickness (AHS), and facilitated more thorough characterisation of their epidemiology. The development of assays based on real-time, reverse-transcription polymerase chain reaction (RT-PCR) to detect and identify the numerous serotypes of BT virus (BTV) and AHS virus (AHSV) has aided in-depth studies of the epidemiology ...
Balasuriya UB.Equine influenza (EI) is a highly contagious disease of horses caused by the equine influenza virus (EIV) H3N8 subtype. EI is the most important respiratory virus infection of horses and can disrupt major equestrian events and cause significant economic losses to the equine industry worldwide. Influenza H3N8 virus spreads rapidly in susceptible horses and can result in very high morbidity within 24-48 h after exposure to the virus. Therefore, rapid and accurate diagnosis of EI is critical for implementation of prevention and control measures to avoid the spread of EIV and to reduce the economi...
Kambayashi Y, Kishi D, Ueno T, Ohta M, Bannai H, Tsujimura K, Kinoshita Y, Nemoto M.Equine coronavirus (ECoV) causes pyrexia, anorexia, lethargy, and sometimes diarrhoea. Infected horses excrete the virus in their faeces, and ECoV is also detected in nasal samples from febrile horses. However, details about ECoV infection sites in the intestinal and respiratory tracts are lacking. To identify the ECoV infection sites in the intestinal and respiratory tracts, we performed an experimental infection study and analysed intestinal and respiratory samples collected from four infected horses at 3, 5, 7, and 14 days post-inoculation (dpi) by real-time reverse transcription polymeras...
Yiğin A.The extensive use of antibacterial agents used for treatment in human and veterinary clinics to increase yield in livestock and aquaculture in developing countries causes the detection and spread of multidrug-resistant (MDR) strains in different sources. The presence of Escherichia coli strains is very common in racehorses and it's a serious problem on horse farms. Extended-spectrum beta-lactamase (ESBL) production is an important source of MDR development in Gram-negative strains such as E.coli. This study aimed to detect the presence of ESBL genes in E. coli strains isolated from horse farms...
Crabtree E, Uribe K, Smith SM, Roberts D, Salmon JH, Bower JJ, Song L, Bastola P, Hirsch ML, Gilger BC.Equine recurrent uveitis (ERU) is a spontaneous, painful, and vision threatening disease affecting up to 25% of equine populations worldwide. Current treatments of ERU are non-specific and have many side effects which limits them to short-term use. In order to develop an effective therapy for ERU, we investigated the use of adeno-associated virus (AAV) gene therapy, exploiting a natural immune tolerance mechanism induced by equine interleukin-10 (Equine-IL10). The purpose of this study was to evaluate the therapeutic efficacy of a single intravitreal (IVT) dose of AAV8-Equine-IL10 gene therapy...
Turlo AJ, Mueller-Breckenridge AJ, Zamboulis DE, Tew SR, Canty-Laird EG, Clegg PD.The main challenge in tendon injury management is suboptimal tissue healing that fails to re-establish original tendon function. Tissue bioengineering is a promising approach for tendon therapy, with potential to improve its functional outcomes. However, evaluation criteria for tissue-engineered tendon are unclear due to the lack of specific markers of differentiated tendon. The study aim was to identify a panel of genes that characterised tendons in comparison to cartilage or muscles and validate those genes, both in human and key species used as models for tendon diseases. Gene expression pr...
Giannetto C, Fazio F, Alberghina D, Giudice E, Piccione G.In mammals, behavioral and physiological processes display 24-hour rhythms that are regulated by the circadian system. In the present study, we investigated clock gene expression in peripheral leukocytes in horses. For this purpose, 10 Italian Saddle gelding horses (9-11 years old; 475 ± 28 Kg) were housed in individual boxes under natural photoperiod and natural environmental temperature. Blood samples were collected at 4-hour intervals over a 48-hour period. The day before the start of sampling, left jugular furrow of each horse was cannulated for the blood sample collection performed in ...
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...
Hue ES, Fortier CI, Laurent AM, Quesnelle YF, Fortier GD, Legrand LJ, Pronost SL.The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 norm. After the development and first validation step, two distinct characterization steps were performed according to the AFNOR norm: (a) characterization of the qRT-PCR assay alone and (b) characterization of the whole analytical method. The validation of the whole analytical method included the portrayal of all steps between the extraction of nucleic acids and the final PCR analysis. Validation of the whole method is very important for vi...
Vissani MA, Tordoya MS, Tsai YL, Lee PA, Shen YH, Lee FC, Wang HT, Parreño V, Barrandeguy M.Equine coital exanthema (ECE) is an infectious, venereally transmitted muco-cutaneous disease affecting mares and stallions, caused by equid alphaherpesvirus 3 (EHV3). Diagnostic tools for rapid identification of EHV3 are of primary importance to diminish the risk of EHV3 dissemination at the time of breeding. In the last years, it has been shown that the performance of the insulated-isothermal polymerase chain reaction (iiPCR) is comparable to virus isolation, nested PCR and real-time PCR (qPCR) in detecting pathogens of various animal species. Analytical sensitivity and specificity of the ii...
Barrandeguy M, Vissani A, Lezica FP, Salamone J, Heguy A, Becerra L, Olguin Perglione C, Thiry E.The temporary disruption of reproductive activities due to equine coital exanthema (ECE), caused by equid herpesvirus 3 (EHV-3), at thoroughbred breeding facilities and embryo transfer centres, has an appreciable economic impact. The aim of the present study was to estimate the prevalence of excretion of EHV-3 in mares without clinical symptoms under field conditions and the re-excretion patterns of the virus in two seropositive (presumably latently infected) mares maintained in isolation for 11 mo. The EHV-3 virus was detected in perineal-vaginal swabs by real time PCR in 14 (6%) of 220 thoro...
Porcellato I, Mecocci S, Mechelli L, Cappelli K, Brachelente C, Pepe M, Orlandi M, Gialletti R, Passeri B, Ferrari A, Modesto P, Ghelardi A....Penile squamous cell carcinomas (SCCs) are common tumors in older horses, with poor prognosis mostly due to local invasion and recurrence. These tumors are thought to be mainly caused by papillomavirus type 2 (EcPV-2). The aim of this study is to characterize the tumor immune environment (TIME) in equine penile tumors. Equine penile epithelial tumors (17 epSCCs; 2 carcinomas , CIS; 1 papilloma, P) were retrospectively selected; immune infiltrate was assessed by histology and immunohistochemistry; RT-qPCR tested the expression of selected chemokines and EcPV-2 DNA and RNA. The results confirme...
Carvalho GM, Ramos CP, Lobato FCF, Guedes RMC, Giaretta PR, Silva ROS.Despite the known importance of Clostridioides (Clostridium) difficile infection (CDI) in animals, there are no published guidelines for the diagnosis of CDI. The performance of the available commercial methods, all standardized for human stool samples, can vary according to the animal species. Thus, the aim of the present study was to review the literature on the detection of C. difficile in pigs, horses, and dogs. The detection of toxins A and B using enzyme immunoassays seems to have low performance in piglet and dog samples, while it shows high sensitivity for the diagnosis of CDI in foal...
Harrington JR, Golding MC, Martens RJ, Halbert ND, Cohen ND.To evaluate a real-time quantitative polymerase chain reaction (QPCR) assay in the detection and quantitation of virulent Rhodococcus equi. Methods: 1 virulent, 2 intermediately virulent, and 2 avirulent strains of R. equi and 16 isolates of bacteria genetically related to R. equi. Methods: The QPCR assay was evaluated for detection and quantitation of the virulence-associated gene (vapA) of R. equi in pure culture and in samples of tracheobronchial fluid, which were inoculated with known numbers of virulent R. equi. Results were compared with those derived via quantitative microbial culture a...
Equine germplasm bank management involves not only the conservation and use of semen doses, in addition it can also be a resource to study stallion semen quality and after thawing semen properties for reproductive purposes. A possible criterion to measure quality may be based on differential gene expression of loci involved during spermatogenesis and sperm quality maturation. The rapid degradation of sperm after thawing affects the integrity and availability of RNA. In this study we have analyzed genes expressed in equine cryopreserved sperm, which provided an adequate amplification, specifici...
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...
Françoso R, Riccio AV, Fernandes CB, Alonso MA, Belli CB.Equine piroplasmosis, disease globally responsible for significant economic losses, is caused by the transmission of the hemoprotozoa Theileria equi and Babesia caballi by ticks. Transplacental transmission has been reported previously in horses, which is not the case for mules´ foals. The present study investigated, by Real Time-PCR (RT-PCR), the presence of both Theileria equi and Babesia caballi in newborn mules before the ingestion of colostrum. Blood samples of 18 mares and their respective mules´ foals were collected and analyzed by RT-PCR. Only one mare was positive for B. caballi. In...
Pearce JW, Galle LE, Kleiboeker SB, Turk JR, Schommer SK, Dubielizig RR, Mitchell WJ, Moore CP, Giuliano EA.Equine recurrent uveitis (ERU) is the most frequent cause of blindness in horses worldwide. Leptospira has been implicated as an etiologic agent in some cases of ERU and has been detected in fresh ocular tissues of affected horses. The objective of this study was to determine the presence of Leptospira antigen and DNA in fixed equine ocular tissues affected with end-stage ERU. Sections of eyes from 30 horses were obtained. Controls included 1) 10 normal equine eyes and 2) 10 equine eyes with a nonrecurrent form of uveitis. The experimental group consisted of 10 eyes diagnosed with ERU based on...
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). ...
Brister H, Barnum SM, Reedy S, Chambers TM, Pusterla N.We validated 2 multiplex real-time PCR (rtPCR) assays based on single nucleotide polymorphisms (SNPs) of the hemagglutinin-1 ( HA1) gene of H3N8 equine influenza A virus (EIV) to determine clade affiliation of prototype and field isolates. Initial validation of the 2 multiplex rtPCR assays (SNP1 and SNP2) was performed using nucleic acid from 14 EIV Florida sublineage clade 1 and 2 prototype strains. We included in our study previously banked EIV rtPCR-positive nasal secretions from 341 horses collected across the United States in 2012-2017 to determine their clade affiliation. All 14 EIV prot...
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...
Gazzonis AL, Morganti G, Porcellato I, Roccabianca P, Avallone G, Gavaudan S, Canonico C, Rigamonti G, Brachelente C, Veronesi F. is a protozoan causing human zoonotic visceral leishmaniasis (ZVL) and visceral-cutaneous canine leishmaniosis (CanL) in the Mediterranean Basin. is able to infect a large number of wild and domestic species, including cats, dogs, and horses. Since the 1990s, clinical cases of equine leishmaniasis (EL), typically characterized by cutaneous forms, have been increasingly diagnosed worldwide. The aim of the present study was to evaluate the presence of clinical forms of EL in CanL-endemic areas in Italy, where exposure of equine populations was ascertained from recent serological surveys. For t...
Cook RF, Barrandeguy M, Lee PA, Tsai CF, Shen YH, Tsai YL, Chang HG, Wang HT, Balasuriya UBR.Control of equine infectious anaemia (EIA) currently depends on serological diagnosis of infected equids. However, recently infected equids may not produce detectable anti-EIAV antibodies up to 157 days post infection and so present a high transmission risk. Therefore, direct nucleic acid detection methods are urgently needed to improve EIAV surveillance and management programs in counties where the disease is endemic. Objective: To evaluate a field-deployable, reverse transcription-insulated isothermal PCR (RT-iiPCR) assay targeting the conserved 5' untranslated region (5' UTR)/exon 1 of the ...
Ghasemi S, Sardari K, Mirshokraei P, Hassanpour H.Application of synthetic matrix metalloproteinases (MMPs) inhibitors, such as doxycycline is one of the possible therapeutic options for osteoarthritis. However, little is known about the protective mechanism of doxycycline in equine models on MMPs inhibitors as well as on serum amyloid A (SAA) gene expression. This study investigated the effects of doxycycline on mRNA expression of MMP-1, MMP-2, MMP-9, MMP-13, and SAA of equine fibroblast-like synoviocytes (FLSs). The FLSs were established from synovial fluids of clinically normal metacarpophalangeal joints of 6 skeletally mature horses. The ...
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 ...
Sauvage AC, Monclin SJ, Elansary M, Hansen P, Grauwels MF.Equine recurrent uveitis (ERU) has been associated with Leptospira spp. infection. No information exists concerning the prevalence of Leptospira-associated ERU in Belgium and about the sensitivity of detection of Leptospira in different ocular media. Objective: To establish the prevalence of intraocular Leptospira spp. in ERU-affected and healthy eyes of horses examined at the Equine Clinic of the University of Liège by real-time PCR and to compare the results of the aqueous and vitreous humour of the same eye. Methods: Cross-sectional. Methods: Sixty-six eyes from 59 client-owned horses with...
Waguespack RW, Kemppainen RJ, Cochran A, Lin HC, Belknap JK.The mediators and signalling cascades important in the initiation of laminitis remain unclear. We therefore wanted to explore the genes and overall signalling mechanisms that play an important role in the developmental stage of laminitis. Objective: To use a broad genomic screening technique to identify novel genes that are differentially regulated in the equine lamellae during the developmental period of laminitis. Methods: Differential mRNA display (DRD) was performed to discover regulated genes, and real-time quantitative polymerase chain reaction (RT-qPCR) was then used to evaluate lamella...
