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.
Boyle AG, Stefanovski D, Rankin SC.Streptococcus equi subsp equi (S. equi) is the cause of "equine strangles" which is a highly infectious upper respiratory disease. Detection of S. equi is influenced by site of specimen collection, method of sampling, and type of diagnostic test that is performed. We hypothesized i) that a loop-mediated isothermal amplification (LAMP) assay that targets the S. equi-specific eqbE gene would be more sensitive than a realtime PCR assay that targets the S. equi-specific seeI gene and ii) that LAMP of specimens obtained by guttural pouch lavage (GPL) would be more sensitive than LAMP of nasopharyng...
Steinbusch MM, Fang Y, Milner PI, Clegg PD, Young DA, Welting TJ, Peffers MJ.The development of effective treatments for the age-related disease osteoarthritis and the ability to predict disease progression has been hampered by the lack of biomarkers able to demonstrate the course of the disease. Profiling the expression patterns of small nucleolar RNAs (snoRNAs) in joint ageing and OA may provide diagnostic biomarkers and therapeutic targets. This study determined expression patterns of snoRNAs in joint ageing and OA and examined them as potential biomarkers. Using SnoRNASeq and real-time quantitative PCR (qRT-PCR) we demonstrate snoRNA expression levels in murine age...
Cymerys J, Słońska A, Brzezicka J, Tucholska A, Chmielewska A, Rola J, Malik P, Bańbura MW.Equine herpesvirus type 1 (EHV-1) causes respiratory infections, abortion and neurological disorders in horses. Molecular epidemiology studies have demonstrated that a single-point mutation in DNA polymerase gene, resulting in an amino acid variation (N752/D752), is significantly associated with the neuropathogenic potential of EHV-1 strains. The aim of the study was to elucidate if there are any differences between neuropathogenic (EHV-1 26) and non-neuropathogenic (Jan-E and Rac-H) EHV-1 strains in their ability to infect neuronal cells. For the tested EHV-1 strains, cytopathic effect (CPE) ...
Kaspar A, Pfister K, Nielsen MK, Silaghi C, Fink H, Scheuerle MC.Strongylus vulgaris has become a rare parasite in Germany during the past 50 years due to the practice of frequent prophylactic anthelmintic therapy. To date, the emerging development of resistance in Cyathostominae and Parascaris spp. to numerous equine anthelmintics has changed deworming management and the frequency of anthelmintic usage. In this regard, reliable detection of parasitic infections, especially of the highly pathogenic S. vulgaris is essential. In the current study, two diagnostic methods for the detection of infections with S. vulgaris were compared and information on the occ...
Balasuriya UB, Lee PA, Tsai YL, Tsai CF, Shen YH, Chang HG, Skillman A, Wang HT, Pronost S, Zhang Y.Equine herpesvirus myeloencephalopathy (EHM), a major problem for the equine industry in the United States, is caused by equine herpesvirus-1 (EHV-1). In addition, EHV-1 is associated with upper respiratory disease, abortion, and chorioretinal lesions in horses. Here we describe the development and evaluation of an inexpensive, user-friendly insulated isothermal PCR (iiPCR) method targeting open reading 30 (ORF30) to detect both neuropathogenic and non-neuropathogenic strains on the field-deployable POCKIT™ device for point-of-need detection of EHV-1. The analytical sensitivity of the EHV-1 ...
Azarpeykan S, Dittmer KE.Housekeeping genes (HKGs) are used as internal controls for normalising and calculating the relative expression of target genes in RT-qPCR experiments. There is no unique universal HKG and HKGs vary among organisms and tissues, so this study aimed to determine the most stably expressed HKGs in the equine kidney. The evaluated HKGs included 18S ribosomal RNA (18S), 28S ribosomal RNA (28S), ribosomal protein L32 (RPL32), β-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase complex (SDHA), zeta polypeptide (YWHAZ), and hypoxanthine phosphoribosyltran...
Vina-Rodriguez A, Eiden M, Keller M, Hinrichs W, Groschup MH. (VEEV) is an from the family that causes epizootic outbreaks in equids and humans in Central and South America. So far, most studies use conventional reverse transcriptase PCR assays for the detection of the different VEEV subtypes. Here we describe the development of a TaqMan quantitative real-time reverse transcriptase PCR assay for the specific detection and quantitation of all VEEV subtypes which uses in parallel a universal equine encephalitis virus control RNA carrying target sequences of the three equine encephalitis viruses. The control RNA was used to generate standard curves for t...
Marth CD, Firestone SM, Glenton LY, Browning GF, Young ND, Krekeler N.Infectious endometritis is a major cause of reduced pregnancy rates in horses. The objectives of this study were to establish a timeline of the innate immune response in the uterus of healthy horses and to investigate the oestrous cycle effect on this. Endometrial biopsies were collected from five horses before and at 3, 12, 24, 48 and 72 h after inoculation of Escherichia coli, once in oestrus and once in dioestrus. They were analysed by quantitative real-time PCR, microbiology and histology. Neutrophil numbers increased from very low levels in the absence of inflammation to severe neutrophi...
Klohonatz KM, Cameron AD, Hergenreder JR, da Silveira JC, Belk AD, Veeramachaneni DN, Bouma GJ, Bruemmer JE.During early pregnancy, the conceptus and mare communicate to establish pregnancy. Cell-secreted vesicles (e.g., exosomes) have been reported in serum. Exosomes contain bioactive materials, such as miRNA, that can mediate cell responses. We hypothesized that a) exosomes are present in mare circulation and quantity varies with pregnancy status, b) exosomes contain miRNAs unique to pregnancy status, and c) miRNAs target pathways in endometrium based upon pregnancy status of the mare. First, serum samples were obtained from mares in a crossover design, with each mare providing samples from a preg...
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...
Fedorka CE, Scoggin KE, Squires EL, Ball BA, Troedsson MHT.The seminal plasma protein, cysteine-rich secretory protein-3 (CRISP-3), has been correlated with increased fertility and first-cycle conception rates, and has been suggested to be involved in the modulation of polymorphonuclear neutrophil and phagocytosis of spermatozoa during the inflammatory response to breeding in the horse. Previous research demonstrated that equine CRISP-3 is located in both the ampulla of the vas deferens and the seminal vesicles. However, this was done with nonquantitative laboratory techniques. In humans and rodents, CRISP-3 has been described as an androgen-dependent...
Azarpeykan S, Dittmer KE, Marshall JC, Perera KC, Gee EK, Acke E, Thompson KG.The aim of this study was to determine the relative abundance and relationship of vitamin D responsive and calcium transporting transcripts (TRPV5, TRPV6, calD9k, calD28k, PMCA, NCX1, CYP27B1, CYP24A1, and VDR) in ovine, canine and, equine kidney using quantitative real-time PCR (RT-qPCR), and then perform a comparison between the three species. Renal tissue samples were harvested post-mortem from 10 horses, 10 sheep, and five dogs. Primers were designed for each gene. For each sample total RNA was extracted, cDNA synthesised, and RT-qPCR was performed. RT-qPCR data were normalised and statist...
Ueno TE, Costa FB, Moraes-Filho J, Agostinho WC, Fernandes WR, Labruna MB.Rickettsia rickettsii is vectored by ticks, and some vertebrate hosts can be sources of infection to ticks during bacteremic periods. In Brazil, the main vector for R. rickettsii is the tick Amblyomma sculptum, a member of the A. cajennense complex. Horses, in turn, are one of the major hosts for A. sculptum. In this study, horses experimentally infected with R. rickettsii were assessed for clinical changes and their capability to transmit the infection to A. sculptum ticks. Four horses were infected with R. rickettsii through either intraperitoneal injection or infestation with R. rickettsii-...
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...
Frank LA, Kania SA, Weyant E.Dermatophilus congolensis causes a crusting dermatitis that affects horses. Diagnosis requires the identification of the organism with cytological evaluation of crust samples. This method can lack sensitivity in chronic cases. Objective: To develop a probe-based real time quantitative polymerase chain reaction (RT-qPCR) test to assist with the diagnosis of dermatophilosis in horses. Methods: Twenty six privately owned horses and seven horses from a research colony were used. Methods: Crust samples, collected from 14 horses with suspected dermatophilosis and 12 horses with crusting skin disease...
Corbin CJ, Legacki EL, Ball BA, Scoggin KE, Stanley SD, Conley AJ.The 5α-reductase enzymes play an important role during male sexual differentiation, and in pregnant females, especially equine species where maintenance relies on 5α-reduced progesterone, 5α-dihydroprogesterone (DHP). Epididymis expresses 5α-reductases but was not studied elaborately in horses. Epididymis from younger and older postpubertal stallions was divided into caput, corpus and cauda and examined for 5α-reductase activity and expression of type 1 and 2 isoforms by quantitative real-time polymerase chain reaction (qPCR). Metabolism of progesterone and testosterone to DHP and dihydro...
Gather T, Walter S, Todt D, Pfaender S, Brown RJP, Postel A, Becher P, Moritz A, Hansmann F, Baumgaertner W, Feige K, Steinmann E, Cavalleri JV.Non-primate hepacivirus (NPHV), a recently discovered hepatotropic virus infecting horses, is phylogenetically the closest known homologue of hepatitis C virus (HCV). The main route for acquiring HCV infection in childhood is vertical transmission. However, nothing is known about the natural mode of transmission for NPHV. To investigate the possibility of vertically transmitted NPHV infection in horses, 20 Thoroughbred broodmares and their foals were monitored during foaling season 2015 until 6 months post-partum. Prepartal serum was taken from the mares, and during foaling umbilical cord bloo...
Köhne M, Ille N, Erber R, Adib Razavi MS, Walter I, Aurich C.Progestin concentration in plasma during the early luteal phase is crucial for endometrial function and conceptus development. We hypothesized that periovulatory gonadotrophin treatment via support of luteal function affects endometrial gene expression in horses. Effect of age was analyzed as well. Shetland mares (n = 8, age 4-25 years) were assigned to the following treatments during five consecutive cycles in alternating order following a cross-over design: treatment hCG/-: preovulatory injection of hCG, but no gonadotrophin injection at detection of ovulation, treatment -/hCG: no preovulat...
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...
de Waal T, Liebenberg D, Venter GJ, Mienie CM, van Hamburg H.African horse sickness (AHS) is an infectious, non-contagious arthropod-borne disease of equids, caused by the African horse sickness virus (AHSV), an orbivirus of the Reoviridae family. It is endemic in sub-Saharan Africa and thought to be the most lethal viral disease of horses. This study focused on detection of AHSV in Culicoides imicola (Diptera: Ceratopogonidae) pools by the application of a RT-qPCR. Midges were fed on AHSV-infected blood. A single blood-engorged female was allocated to pools of unfed nulliparous female midges. Pool sizes varied from 1 to 200. RNA was extracted and prepa...
Klein C."Maternal recognition of pregnancy" (MRP) is commonly used to describe the ongoing embryo-maternal communication during early pregnancy that culminates in prevention of luteolysis and ensures ongoing progestin support. The conceptus-derived pregnancy recognition signal has not yet been identified in the mare. Although equine conceptuses produce substantial amounts of estrogens, there is a lack of evidence that estrogens are the pregnancy recognition signal in mares. Conceptus mobility is integral to MRP and is driven by conceptus-derived prostaglandin production. Cessation of conceptus mobilit...
Orsztynowicz M, Pawlak P, Podstawski Z, Nizanski W, Partyka A, Gotowiecka M, Kosiniak-Kamysz K, Lechniak D.Predicting male fertility on non-invasive sperm traits is of big importance to human and animal reproduction strategies. Combining the wide range of parameters monitored by computer-assisted sperm analysis (CASA) with some molecular traits (e.g. mtDNA content) may help to identify markers of the male fertility. The aim of this study was to characterize variation in the mtDNA copy number in equine sperm and to investigate whether mtDNA content is correlated with quality traits of stallion spermatozoa and the age of the male. Ejaculates collected from 53 fertile stallions were divided into four ...
Carossino M, Lee PY, Nam B, Skillman A, Shuck KM, Timoney PJ, Tsai YL, Ma LJ, Chang HF, Wang HT, Balasuriya UB.Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory and reproductive disease of horses. Most importantly, EAV induces abortion in pregnant mares and can establish persistent infection in up to 10-70% of the infected stallions, which will continue to shed the virus in their semen. The objective of this study was to develop and evaluate a reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) for the detection of EAV in semen and tissue samples. The newly developed assay had a limit of detection of 10 RNA copies and a 10-fol...
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...
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...
