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.
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...
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...
Colahan PT, Kollias-Bakert C, Leutenegger CM, Jones JH.Physical exertion is a stimulus for the upregulation of cytokine production including IL-1beta, IL-1ra, IL-2, IL-4, IL-6, IL-10 and TNF-alpha in horses. To investigate that hypothesis, we initiated training of 5 stall-rested Thoroughbreds. Blood samples were drawn before and weekly during training. The relative transcription of mRNA within the leucocytes was measured using real time TaqMan quantitative PCR. The training protocol was walking (3 min), trotting (3 min) and cantering/galloping (6 min) increasing in intensity weekly (6 to 12 m/s) and culminating in an intense exercise period. Compa...
Silva MA, Silva KM, Jesus AL, Barros LO, Corteggio A, Altamura G, Borzacchiello G, Freitas AC.Papillomavirus (PV) are double-stranded DNA viruses that can cause both benignant and malignant tumours in mammals. Twelve genotypes of bovine papillomavirus (BPV1-12) have been identified so far. The presence of BPV1 and 2 has been found in the body fluids of cattle and horses. The aim of this study is to investigate the presence of BPV DNA and the expression of viral genes in the blood and sperm cells of healthy horses using PCR and RT-PCR. BPV-1 or 2 was detected in 14 of 70 blood samples (20%) and in 11 of 31 semen samples (35%). In five of fourteen blood samples, the E5 expression tested ...
Miragliotta V, Pirone A, Donadio E, Abramo F, Ricciardi MP, Theoret CL.Convincing evidence shows that persistent or excessive expression of osteopontin (OPN) is linked to fibroproliferation of various organs in laboratory animals and in man, such that its downregulation is a logical therapeutic objective. Objective: To investigate OPN expression in an equine model of wound healing and in clinical specimens of equine exuberant granulation tissue and human keloids in an effort to better understand the contribution of this protein to inflammation-associated skin fibrosis. Methods: Description of gene and protein expression in an experimental equine model of wound he...
Aklilu N, Batten C, Gelaye E, Jenberie S, Ayelet G, Wilson A, Belay A, Asfaw Y, Oura C, Maan S, Bachanek-Bankowska K, Mertens PP.African horse sickness (AHS) is associated with high morbidity and mortality in equids, especially horses. A retrospective analysis was carried out concerning 737 AHS outbreaks that occurred during 2007-2010 in Ethiopia. A total of ten outbreaks were investigated in the study period. All four forms of the disease (pulmonary, cardiac, horse sickness fever and the combined form) were observed, with the cardiac form being the most prevalent. Multiple African horse sickness virus serotypes (AHSV-2, AHSV-4, AHSV-6, AHSV-8 and AHSV-9) were detected by molecular methods (type-specific real-time RT-PC...
North SE, Wakeley PR, Mayo N, Mayers J, Sawyer J.Infection with Streptococcus equi subspecies equi (S. equi) is endemic in the UK. A proportion of horses serve as long-term carriers and act as a reservoir of infection. Detection of these persistently infected horses is difficult using standard culture techniques owing to a lack of sensitivity and overgrowth by contaminating bacteria. In addition, differentiation of this causative bacterium from the closely related S. equi zooepidemicus has made the development of reliable and accurate diagnostic tests difficult. Objective: To develop and validate a sensitive and specific real-time PCR assay ...
Johnson AL, Sweeney RW, McAdams SC, Whitlock RH.Clostridium botulinum type B is estimated to cause more than 85% of cases of equine botulism in the United States, as well as many outbreaks in cattle. In this study, a quantitative real-time polymerase chain reaction for detection of the neurotoxin gene of C. botulinum type B was compared to the mouse bioassay using 45 positive and 43 negative samples of equine, bovine or associated environmental origin. The sensitivity of the qPCR assay was 96%, whereas the sensitivity of the mouse bioassay was 84%. The specificity of the qPCR assay was 95% and the specificity of the mouse bioassay was 100%....
Erol E, Jackson CB, Steinman M, Meares K, Donahoe J, Kelly N, Locke S, Smith JL, Carter CN.A comprehensive evaluation of the real-time PCR assay for leptospirosis in comparison with other diagnostic assays on a large-scale basis is fundamental in validating the assay and determining the causes of equine abortions. Objective: To compare and evaluate the diagnostic value of real-time PCR assay for leptospirosis with traditional methods in equine leptospiral abortions. Methods: Cross-sectional observational study. Methods: A Leptospira spp. fluorescent antibody test (FAT), microscopic agglutination test (MAT) and real-time PCR (targeting the LipL32 gene) were compared and evaluated in ...
Riihimäki M, Raine A, Pourazar J, Sandström T, Art T, Lekeux P, Couëtil L, Pringle J.The aim of this study was to evaluate the contribution of bronchial epithelium to airway inflammation, with focus on mRNA and protein expression of cytokines of innate immunity IL-6, IL-10 and TNF-alpha, in horses with Recurrent Airway Obstruction (RAO) during exacerbation and in remission. Results: Despite marked clinical and physiologic alterations between exacerbation and after remission in the RAO horses no differences were detected in either cytokine mRNA or protein levels. Moreover, the expression of investigated cytokines in RAO horses on pasture did not differ from controls. In compari...
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...
Magdesian KG, Leutenegger CM.Clostridium difficile infection can occur in the dams of sick foals, but it is unknown if mares and foals share the same isolates. In this study, C. difficile isolates from fecal samples of 11 mares paired with 11 foals were genotyped by arbitrarily primed PCR; two mares and three foals in five mare-foal pairs had diarrhea. Fecal immunoassays were utilized to detect C. difficile common antigen and toxin A. Quantitative real-time PCR (qPCR) systems were developed to detect genes for toxins A and B, as well as for binary toxin B. Sequences of all toxins were present in all isolates, although onl...
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...
Pereira GR, Lorenzo PL, Carneiro GF, Ball BA, Bilodeau-Goeseels S, Kastelic J, Pegoraro LM, Pimentel CA, Esteller-Vico A, Illera JC, Granado GS....The objectives of this study were to evaluate the effects of equine growth hormone (eGH) on nuclear and cytoplasmic maturation of equine oocytes in vitro, steroid production by cumulus cells, and expression and subcellular localization of eGH-receptors (eGH-R) on equine ovarian follicles. Cumulus-oocyte complexes (COCs) were recovered by aspirating follicles <30 mm in diameter from abattoir-derived ovaries. The COCs were morphologically evaluated and randomly allocated to be cultured in either a control maturation medium or supplemented with 400 ng/mL eGH, for 30 h at 38.5°C in air with 5%...
Zakhia R, Dupuis AP, Khodr F, Fadel M, Kramer LD, Haddad N.West Nile virus (WNV) has never been reported from Lebanon. Yet, this country is located on the flyway of migratory birds in the Middle East region. Serological screening was conducted to assess the potential circulation of this virus. Human, horse, and chicken sera were collected from the Bekaa and North districts. Specific IgG and IgY were first screened by ELISA. Then, positive samples were confirmed by plaque reduction neutralization test (PRNT). Besides this, adult mosquitoes were collected and tested for the presence of WNV RNA using conventional RT-PCR. Sera screening revealed a seropre...
Pecoraro HL, Felippe MJB, Miller AD, Divers TJ, Simpson KW, Guyer KM, Duhamel GE.Common variable immunodeficiency (CVID) is a rare condition in adult horses characterized by hypogammaglobulinemia and increased susceptibility to parasitic and bacterial infections, including recurrent respiratory diseases, septicemia, and meningitis. Lyme disease is often included as a differential diagnosis in CVID horses with signs of meningitis; however, the Borrelia burgdorferi organism has not been demonstrated previously within central nervous system tissues of CVID horses with neurologic disease, to our knowledge. We report herein a case of neuroborreliosis in a CVID horse, confirmed ...
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...
Van Eeden C, Zaayman D, Venter M.Recently Shuni virus (SHUV) has been identified in clinical cases of neurological disease in horses in South Africa. Being that it was one of the less recognized orthobunyaviruses, with limited clinical descriptions of disease dating back to the 1960s and 1970s, SHUV-specific assays were never developed. In this study, the development of a nested real-time PCR assay is described for the detection of SHUV by means of melt-curve analysis using fluorescence resonance energy transfer (FRET) probe technology. The assay was validated against previously positive clinical specimens and a dilution seri...
Bannai H, Takahashi Y, Ohmura H, Ebisuda Y, Mukai K, Kambayashi Y, Nemoto M, Tsujimura K, Ohta M, Raidal S, Padalino B.This study evaluated the effects of 12-hour transportation on immune responses to equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4). Possible replication of EHV-1 and EHV-4 was monitored by real-time PCR of nasal swabs and peripheral blood mononuclear cells (PBMCs), and changes in systemic and mucosal antibodies were investigated. Six healthy Thoroughbreds with transport experience were transported in commercial trucks, repeating the same three-hour route four times. Blood samples for cortisol measurement were taken before departure and every three hours. Nasal swabs, PBMCs, nasal wash and ...
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...
Gilger BC, Salmon JH, Yi NY, Barden CA, Chandler HL, Wendt JA, Colitz CM.To determine the role of intraocular bacteria in the pathogenesis of equine recurrent uveitis (ERU) in horses from the southeastern United States by evaluating affected eyes of horses with ERU for bacterial DNA and intraocular production of antibodies against Leptospira spp. Methods: Aqueous humor, vitreous humor, and serum samples of 24 clinically normal horses, 52 horses with ERU, and 17 horses with ocular inflammation not associated with ERU (ie, non-ERU inflammation). Methods: Ribosomal RNA quantitative PCR (real-time PCR) assay was used to detect bacterial DNA in aqueous humor and vitreou...
Riihimäki M, Lilliehöök I, Raine A, Berg M, Pringle J.The aim of this study was to assess clinical signs and altered pulmonary cell expression of cytokines related to eosinophil kinetics in horses with pulmonary eosinophilia. Pulmonary eosinophilia was detected by bronchoalveolar lavage (BAL) in a group of standardbreds in training. Horses had detailed clinical examination, bronchoscopy, endobronchial biopsy and BAL on three occasions at approximately 6 month intervals. During the second sampling period BAL eosinophils were significantly elevated (p>0.010), with five horses having from 5% to 37% eosinophils in BAL. Neither detailed clinical ex...
Staji H, Yousefi M, Hamedani MA, Tamai IA, Khaligh SG.Anaplasma spp. are among the most recognized arthropod-borne infectious agents. Although the novel A. capra has been isolated from wildlife, livestock, and hard ticks from many parts of the world, there is no report regarding the identification of this pathogen from equines and little is known about the epidemiology of A. capra in Equidae. In this study, A. capra was identified in two out of ten blood specimens of wild onagers (Equus hemionus onager) during a routine health check-up in Semnan, Iran by light microscopy and molecular analyses while other pathogens were not detected. First, inclu...
Murphy BA, Vick MM, Sessions DR, Cook RF, Fitzgerald BP.The master mammalian pacemaker in the brain controls numerous diverse physiological and behavioral processes throughout the organism. Timing information is continually transmitted from the master clock to peripheral organs to synchronize rhythmic daily oscillations of clock gene transcripts and control local physiology. To investigate the presence of peripheral clocks in the horse, quantitative real-time RT-PCR assays were designed to detect levels of equine clock genes. Expression profiles for Per2, Bmal1 and Cry1 were first determined in a synchronized equine cell line. Subsequently, express...
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...
Carossino M, Balasuriya UBR, Thieulent CJ, Barrandeguy ME, Vissani MA, Parreño V.Equine rotavirus A (ERVA) is the leading cause of diarrhea in foals, with G3P[12] and G14P[12] genotypes being the most prevalent. Recently, equine G3-like RVA was recognized as an emerging infection in children, and a group B equine rotavirus (ERVB) was identified as an emergent cause of foal diarrhea in the US. Thus, there is a need to adapt molecular diagnostic tools for improved detection and surveillance to identify emerging strains, understand their molecular epidemiology, and inform future vaccine development. We developed a quadruplex TaqMan RT-qPCR assay for differentiation of ERVA an...
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...
