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.
Gold JR, Perkins GA, Erb HN, Ainsworth DM.Septicemia initiates the production of pro-inflammatory (interleukin [IL] 1-beta [IL-1beta], interferon-gamma [IFN-gamma], IL-6), and anti-inflammatory (IL-4) cytokines. The transcription of some of these proteins (IL-8, IL-6) is linked to endotoxin-induced activation of the toll-like receptor 4 (TLR4) on peripheral blood mononuclear cells (PBMC). Objective: Septic foals fail to increase gene expression of IFN-gamma. Nonsurviving septic foals exhibit distinctive cytokine profiles. Methods: Twenty-one septic and 20 healthy neonatal foals. Methods: Using real-time polymerase chain reaction, gene...
Huang H, Lavoie-Lamoureux A, Moran K, Lavoie JP.Little is known concerning the possible contribution of T helper 2 (Th2)-type cytokines to the recruitment of neutrophils into the lung tissue. In the present study, endothelial cells from equine pulmonary arteries were cultured in the presence of recombinant equine (re) IL-4 and reIL-5, and the cytokine mRNA expression of molecules implicated in the chemotaxis and migration of neutrophils was studied using real-time RT-PCR. The functional response of reIL-4-induced endothelial cell stimulation on neutrophil migration was also studied using a chemotaxis chamber. ReIL-4 either increased the exp...
Quinlivan M, Cook RF, Cullinane A.In 2006, an outbreak of equine infectious anaemia (EIA) occurred in Ireland. The initial source of the outbreak is believed to have been contaminated plasma imported from Italy. This paper presents the nucleotide sequence of the gag gene of the virus identified in Ireland (EIAV(Ire)), the first for a European strain of EIAV. Comparison of the gag gene with North American and Asian strains of the virus showed that the gag gene is less well conserved than previously believed, and that EIAV strains can have similar phenotypes despite considerable variations in genotype. On the basis of the deduce...
Fumuso EA, Aguilar J, Giguère S, Rivulgo M, Wade J, Rogan D.Our objective was to characterize immune parameters in susceptible (SM) and resistant (RM) mares, with and without artificial insemination (AI) and immunomodulation. Eight RM and eight SM were selected based on their reproductive history and functional tests. Both groups of mares were evaluated during three consecutive cycles: Cycle 1, untreated cycle (control); Cycle 2, AI with dead semen; Cycle 3, AI with dead semen and immunomodulation. Endometrial biopsies were taken during the three cycles as follows: Cycle 1--at estrus, when follicles > or =35mm and at diestrus (7+/-1 days after ovulatio...
David F, Farley J, Huang H, Lavoie JP, Laverty S.To evaluate mRNA expression of several proinflammatory and anti-inflammatory cytokines and chemokines in equine unstimulated and interleukin-1beta (IL-1beta)-stimulated chondrocytes. Methods: In vitro experiment using equine chondrocyte cultures. Methods: Whole articular cartilage from metacarpophalangeal joints (n=5 horses; 10 fetlocks). Methods: Chondrocyte monolayer cultures were established from digested adult equine articular cartilage and stimulated with 5 ng/mL of recombinant human IL-1beta. RNA was extracted from the cells 24 hours after stimulation. IL-1beta, IL-4, IL-6, IL-8, tumor n...
Buling A, Criado-Fornelio A, Asenzo G, Benitez D, Barba-Carretero JC, Florin-Christensen M.The haemoparasites Babesia bovis and Babesia bigemina affect cattle over vast areas of the tropics and temperate parts of the world. Microscopic examination of blood smears allows the detection of clinical cases of babesiosis, but this procedure lacks sensitivity when parasitaemia levels are low. In addition, differentiating between similar haemoparasites can be very difficult. Molecular diagnostic procedures can, however, overcome these problems. This paper reports a quantitative PCR (qPCR) assay involving the use of SYBR Green. Based on the amplification of a small fragment of the cytochrome...
Lindbloom SM, Farmerie TA, Clay CM, Seidel GE, Carnevale EM.Human chorionic gonadotropin (hCG) was administered to mares in estrus with large, dominant ovarian follicles to initiate follicular and oocyte maturation. Follicular contents were collected at 0, 2, 4 and 6 h after hCG. Epiregulin, amphiregulin and phosphodiesterase (PDE) mRNA contents of granulosa cells (PDE 4D) were determined by reverse transcription and real-time PCR; PDE 3A mRNA content of single oocytes was determined similarly. Copy numbers of mRNA did not increase for PDE 3A or 4D over the time interval studied. Amounts of epiregulin and amphiregulin mRNA were correlated (r=0.98) when...
Båverud V, Johansson SK, Aspan A.Strangles is a contagious equine disease caused by Streptococcus equi subsp. equi. In this study, clinical strains of S. equi (n=24) and Streptococcus equi subsp. zooepidemicus (n=24) were genetically characterized by sequencing of the 16S rRNA and sodA genes in order to devise a real-time PCR system that can detect S. equi and S. zooepidemicus and distinguish between them. Sequencing demonstrated that all S. equi strains had the same 16S rRNA sequence, whereas S. zooepidemicus strains could be divided into subgroups. One of these (n=12 strains) had 16S rRNA sequences almost identical with the...
Wensman JJ, Thorén P, Hakhverdyan M, Belák S, Berg M.Borna disease virus (BDV) is a non-segmented, negative-stranded RNA virus, which infects cells of the central nervous system (CNS) in many different species. BDV is the causative agent of the neurological disorders in horses and sheep termed classical Borna disease (BD), as well as staggering disease in cats. At present, the diagnosis staggering disease or feline BD is made by histopathology or immunohistochemistry of the CNS. In order to obtain a better clinical diagnostic tool, a duplex real-time RT-PCR assay (rRT-PCR) was developed. TaqMan probes and primers specific for the BDV P and BDV L...
Diallo IS, Hewitson G, Wright LL, Kelly MA, Rodwell BJ, Corney BG.A multiplex real-time PCR was designed to detect and differentiate equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). The PCR targets the glycoprotein B gene of EHV-1 and EHV-4. Primers and probes were specific to each equine herpesvirus type and can be used in monoplex or multiplex PCRs, allowing the differentiation of these two closely related members of the Alphaherpesvirinae. The two probes were minor-groove binding probes (MGB) labelled with 6-carboxy-fluorescein (FAM) and VIC for detection of EHV-1 and EHV-4, respectively. Ten EHV-1 isolates, six EHV-1 positive clinical samples...
Belknap JK, Giguère S, Pettigrew A, Cochran AM, Van Eps AW, Pollitt CC.Recent research has indicated that inflammation plays a role in the early stages of laminitis and that, similar to organ failure in human sepsis, early inflammatory mechanisms may lead to downstream events resulting in lamellar failure. Characterisation of the type of immune response (i.e. innate vs. adaptive) is essential in order to develop therapeutic strategies to counteract these deleterious events. Objective: To quantitate gene expression of pro-inflammatory cytokines known to be important in the innate and adaptive immune response during the early stages of laminitis, using both the bla...
Anderson ME, Weese JS.Screening for nasal colonization is an important aspect of many methicillin-resistant Staphylococcus aureus (MRSA) control programs. Real-time polymerase chain reaction (RT-PCR) is an attractive alternative to standard culture techniques because of the considerably shorter turnaround time. An assay has been validated for diagnostic purposes in humans, however this methodology has not been evaluated in horses. The purpose of this study was to compare an RT-PCR assay for rapid identification of MRSA directly from nasal swabs in horses to standard culture techniques. Nasal swabs collected from 29...
Kolm G, Knapp E, Wagner R, Klein D.Lactoferrin (LF), a glycogen of the transferrin family with anti-bacterial and immunomodulatory properties, is expressed in various secretions and tissues. Cutaneous LF serves as a mast cell stabilising compound, modulates T cell activity and is found during IgE-mediated late phase reactions at allergen challenged sites. Culicoides hypersensitivity (CHS) in horses is a common IgE-mediated allergic dermatitis, characterised by an early and late phase cutaneous reaction upon allergen challenge. The aim of the study presented here was to examine whether LF mRNA expression in skin biopsies from ho...
Schneeweiss W, Stanek C, Wagner M, Hein I.The potential of five different commercial DNA isolation methods to remove real-time PCR inhibitors from the synovial fluid of horses, cattle and pigs was investigated. All kits with the exception of one included a silica column-based purification of the DNA. With the fifth kit, DNA purification is achieved by removing contaminating macromolecules by a desalting process. We used a recently developed method based on comparison of the real-time PCR signal of an artificial target incorporated into each PCR reaction in the presence of the isolated DNA from the sample, and in control samples contai...
Yuan Z, Gallagher A, Gault EA, Campo MS, Nasir L.Bovine papillomavirus (BPV) type 2 is involved in carcinogenesis of the urinary bladder in cattle, while BPV-1 is commonly associated with equine sarcoid tumours. In both cases the early viral proteins are expressed, but virion is not produced. Given the similarities in BPV biology between the tumours in cattle and horses, bovine bladder cancers and equine sarcoids were compared with respect to physical status, load of viral DNA and variability of the E5 open reading frame (ORF). Rolling circle amplification demonstrated that BPV-1 and BPV-2 genomes exist as double stranded, episomal, circular...
van den Hoven R, Duvigneau JC, Hartl RT, Gemeiner M.On four occasions, four horses with heaves and four horses with small airway inflammatory diseases inhaled 0.9% saline based aerosol mixtures with or without lipopolysaccharides (LPS). Prior to the first saline and LPS inhalation, horses were untreated, while three and a half days prior to the third and forth inhalation horses had received 0.8 microg/kg clenbuterol intravenously twice daily. The messenger RNA (mRNA) expression of tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-4, IL-6, IL-8, IL-10 and interferon- gamma (IFN- gamma) was investigated by RT-PCR, all of which ...
Southwood LL, Lindermann J, Zarucco L, Driessen B, Olander H, Habecker P, Birks EK, Frisbie DD.Growth factors (GF) are important for maintenance and repair of intestinal mucosal structure and function, but there have been no studies investigating growth factor (GF) or growth factor receptor (GF-R) mRNA expression in the intestine of horses with large colon volvulus (LCV). Objective: (1) To determine mRNA expression for epidermal growth factor (EGF), EGF receptor (EGF-R), insulin-like growth factor-I (IGF), IGF receptor (IGF-R), vascular endothelial growth factor (VEGF) and VEGF receptor (VEGF-R) in the intestine of horses with an LCV compared to normal intestine. (2) To measure the corr...
Pusterla N, Wilson WD, Conrad PA, Barr BC, Ferraro GL, Daft BM, Leutenegger CM.This study was designed to determine the relative levels of gene transcription of selected pathogens and cytokines in the brain and spinal cord of 12 horses with equine protozoal myeloencephalitis (EPM), 11 with equine herpesvirus type 1 (EHV-1) myeloencephalopathy, and 12 healthy control horses by applying a real time pcr to the formalin-fixed and paraffin-embedded tissues. Total rna was extracted from each tissue, transcribed to complementary dna (cDNA) and assayed for Sarcocystis neurona, Neospora hughesi, EHV-1, equine GAPDH (housekeeping gene), tumour necrosis factor (TNF)-alpha, interfer...
Kolm G, Klein D, Knapp E, Watanabe K, Walter I.Lactoferrin (LF) is an estrogen-regulated glycoprotein with well-described antibacterial and immunomodulatory properties. The present study is the first report on LF expression in horse endometrial specimens. Mares chosen for the study were either resistant or susceptible for persisting mating-induced endometritis (PMIE) during the natural ovulatory cycle and in early pregnancy. Our investigations included immunostaining for LF protein and CD18, a leukocyte marker, as neutrophils are a possible source for LF in the endometrium. Quantification of LF mRNA was performed by use of real-time RT-PCR...
Schindler AR, Vögtlin A, Hilbe M, Puorger M, Zlinszky K, Ackermann M, Ehrensperger F.Borna disease is a severe, immunopathological disorder of the central nervous system caused by the infection with the Borna disease virus (BDV). The detection of BDV in diseased animals, mainly sheep and horses, is achieved by histological, immunohistochemical and serological approaches and/or PCR-based technologies. In the present study, reverse transcription, real-time PCR assays were established for the detection of BDV in the brain tissue from sheep and horses, using loci for the p40 (nucleoprotein) and the p24 (phosphoprotein) genes. The PCRs were equally specific and sensitive, detecting...
Hussey SB, Clark R, Lunn KF, Breathnach C, Soboll G, Whalley JM, Lunn DP.Equine herpesvirus-1 (EHV-1) infection is common in young horses throughout the world, resulting in respiratory disease, epidemic abortion, sporadic myelitis, or latent infections. To improve on conventional diagnostic tests for EHV-1, a real-time polymerase chain reaction (PCR) technique was developed, using primers and probes specific for the EHV-1 gB gene. Amplification efficiencies of 100% +/- 5% were obtained for DNA isolated from a plasmid, infected peripheral blood mononuclear cells (PBMCs), and nasal secretions from infected ponies. The dynamic range of the assay was 8 log10 dilutions,...
Pusterla N, Wilson WD, Conrad PA, Mapes S, Leutenegger CM.To determine gene transcription for cytokines in nucleated cells in CSF of horses without neurologic signs or with cervical stenotic myelopathy (CSM), West Nile virus (WNV) encephalitis, equine protozoal myeloencephalitis (EPM), or spinal cord trauma. Methods: 41 horses (no neurologic signs [n = 12], CSM [8], WNV encephalitis [9], EPM [6], and spinal cord trauma [6]). Methods: Total RNA was extracted from nucleated cells and converted into cDNA. Gene expression was measured by use of real-time PCR assay and final quantitation via the comparative threshold cycle method. Results: Cytokine genes ...
Stadejek T, Mittelholzer Ch, Oleksiewicz MB, Paweska J, Belák S.Equine arteritis virus (EAV) was detected by RT-nested PCR in semen samples from a naturally infected South African donkey. Sequence analysis of the amplified ORF5 fragment revealed only 60 to 70% nucleotide identity to a panel of EAV reference sequences. The unique donkey EAV sequence was also found to be stable during passage in horses. The sequence data reported in this study indicate that the South African donkey variant might represent a new genotype of EAV. The distinct genetic properties of the South African asinine strain of EAV suggest a divergent evolution of this arterivirus in vari...
Wang Y, Zhang XY, Wei LL, Wu DL, Wang XJ, Xiang WH, Shen RX, Shao YM.To evaluate the relationship between the transcriptional level of IFN-gamma mRNA in peripheral blood mononuclear cells (PBMC) and immune protective response driven by inoculated horses with donkey leukocyte attenuated vaccine of EIAV(DLV), and to elucidate the immune mechanism of DLV. Methods: A real-time PCR method was established for quantitative detection of IFN-gamma mRNA level from horse PBMCs. Twelve horses were divided into vaccination group, healthy control group, challenging control group and EIAV natural infection group. The transcriptional level and distribution of IFN-gamma mRNA in...
Semevolos SA, Nixon AJ, Fortier LA, Strassheim ML, Haupt J.The purpose of this study was to determine changes in the expression of regulatory molecules in normal equine articular cartilage throughout development up to 18 months of age. The hypothesis was that expression of these regulatory molecules would decrease from birth to postpubescence. Cartilage was harvested from normal femoropatellar or scapulohumeral joints from 34 fresh horse cadavers. Horses were placed in four age groups [prenatal (n = 5); prepubertal, 0-6 months (n = 11); pubertal, 7-14 months (n = 13); and postpubertal, 15-18 months (n = 5)]. Indian hedgehog (Ihh), Gli1, Gli3, Patched1...
Kolm G, Knapp E, Wagner R, Klein D.Interleukin-1beta (IL-1beta) is a primary cytokine of the skin that has a pivotal role in keratinocyte differentiation, epidermal wound healing and host defense. Pathological increase of cutaneous IL-1beta is associated with edema formation, epidermal hyperproliferation and atopic dermatitis in humans. However, in horses the role of cutaneous IL-1beta in edema formation and allergic skin disease has not been characterised so far. Particularly in Culicoides hypersensitivity (CHS), intradermal injection of Culicoides extract may be associated with enhanced transcription of local IL-1beta. To exa...
Rodríguez-Lázaro D, Lewis DA, Ocampo-Sosa AA, Fogarty U, Makrai L, Navas J, Scortti M, Hernández M, Vázquez-Boland JA.We developed a novel quantitative real-time PCR (Q-PCR) method for the soil actinomycete Rhodococcus equi, an important horse pathogen and emerging human pathogen. Species-specific quantification was achieved by targeting the chromosomal monocopy gene choE, universally conserved in R. equi. The choE Q-PCR included an internal amplification control (IAC) for identification of false negatives. A second Q-PCR targeted the virulence plasmid gene vapA, carried by most horse isolates but infrequently found in isolates from other sources. The choE-IAC and vapA assays were 100% sensitive and specific ...
Gregg AJ, Fortier LA, Mohammed HO, Mayr KG, Miller BJ, Haupt JL.To evaluate the effects of interleukin (IL)-1beta on proteoglycan metabolism in equine cartilage explants when cultured in the presence of synoviocytes. Methods: Samples of cartilage and synovium collected from the femoropatellar joints of three 2- to 3-year-old horses. Methods: 3 experimental groups were established: cartilage explants only, synoviocytes only, and cartilage explants-synoviocytes in coculture. In each group, samples were cultured with or without IL-1beta (10 ng/mL) for 96 hours. Glycosaminoglycan (GAG) content of cartilage and medium samples was measured by use of a spectropho...
Pusterla N, Magdesian KG, Mapes S, Leutenegger CM.To determine gene expression of selected molecular markers (tumor necrosis factor [TNF]-alpha, interleukin [IL]-1beta, IL-6, IL-8, IL-10, procalcitonin [PCT], and transforming growth factor [TGF]-beta) in the blood of healthy and sick foals. Methods: 28 sick foals without sepsis, 21 foals with sepsis, and 21 healthy foals. Methods: Total RNA was extracted from blood samples and converted into complementary DNA (cDNA). Gene expression was measured for the molecular markers by use of real-time PCR assay, and final quantitation was performed with the comparative threshold cycle method. Results: S...
Allen GP, Breathnach CC.Neurological disease in horses caused by infection with certain 'paralytic' strains of equine herpesvirus-1 (EHV-1) is a potentially devastating condition the pathogenesis of which is poorly understood. Preliminary observations in both experimentally induced and naturally occurring cases of the central nervous system disease have revealed a more robust cell-associated viraemia in horses infected with paralytic isolates of EHV-1, relative to horses infected with abortigenic isolates. To investigate further this pathogenesis-relevant question, the present study was performed using a greater numb...
Zoll WM, Prakoso D, Dark M, Liu J, Stockdale-Walden H, Long MT.Eosinophils within the central nervous system are abnormal and are usually associated with fungal or parasitic infections in horses. Causative agents include Halicephalobus gingivalis, Sarcocystis neurona, and Neospora hughesi. Confirmation of these organisms via specific testing is typically not performed, and final diagnoses are often presumptive. With molecular technology, many of these organisms can now be confirmed. This is important for emerging and zoonotic pathogens, including Angiostrongylus cantonensis, an emerging parasite of interest in the southeastern United States. We retrospect...
Solinger N, Sonea IM.Tachykinins have profound effects on equine intestinal motility, but the distribution of the neurokinin receptors (NKRs) through which they act is unknown. This study reports the distribution of one of these receptors, the neurokinin-1 receptor (NK1R), in smooth muscle throughout the equine intestinal tract. Objective: To quantify the distribution of the NK1R, based upon mRNA expression, in smooth muscle of different regions of the equine intestinal tract. Methods: Nine regions of the intestinal tract were sampled in 5 mature horses. Total RNA was isolated from smooth muscle and reverse transc...
Packer MJ, German AJ, Hunter L, Trayhurn P, Proudman CJ.Adipose tissue is an important source of inflammatory cytokines (adipokines) and adiposity has been identified as having a significant effect on human morbidity and mortality. Obesity is also an emerging welfare problem in the UK horse population, but the role that it plays in secondary diseases is unclear. Objective: To examine the expression of inflammation-related adipokine genes in retroperitoneal adipose tissue of horses undergoing emergency abdominal surgery and to explore associations with adiposity and post operative survival. Methods: Retroperitoneal adipose tissue samples were obtain...
Rowe Á, Flanagan S, Barry G, Katz LM, Lane EA, Duggan V.Warmblood Fragile Foal Syndrome (WFFS) is an autosomal recessive disorder caused by a mutation in the procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1) gene. Homozygosity for the mutation results in defective collagen synthesis which clinically manifests as the birth of non viable or still born foals with abnormally fragile skin. While the mutation has been identified in non Warmblood breeds including the Thoroughbred, to date all homozygous clinically affected cases reported in the scientific literature are Warmblood foals. The objective of this study was to investigate the carrier f...
Léon A, Versmisse Y, Despois L, Castagnet S, Gracieux P, Blanchard B.Isolation and identification of Taylorella equigenitalis, the causative agent of contagious equine metritis, by bacteriology is laborious and does not permit differentiation from the other member of the genus, Taylorella asinigenitalis. Moreover, other organisms such as Klebsiella pneumoniae and Pseudomonas aeruginosa can also cause endometritis in mares and warrant diagnostic detection. Our objectives were to develop a rapid preparation method for field swab samples and to validate this protocol using new multiplex real-time polymerase chain reaction (rtPCR) detection tools for identification...
Sykes BW, Furr M, Giguère S.Twelve healthy horses were assigned to treatment or control groups. Treated horses received PGG-Glucan[ED-1] (1 mg/kg, IV) 24 hours prior to peripheral blood mononuclear cell (PBMC) isolation. PBMCs were isolated and incubated in the presence of lipopolysachharide (LPS). At 0, 6, 12, 24 and 48 hours messenger RNA (mRNA) was extracted. Reverse transcription polymerase chain reaction (PCR) was performed and cytokine mRNA expression for tumor necrosis factor alpha (TNFalpha), interleukin 1beta (IL-1beta), interleukin 10 (IL-10) and interferon gamma (IFN-gamma) determined using real time PCR. A si...
Kinoshita Y, Kakoi H, Ishige T, Yamanaka T, Niwa H, Uchida-Fujii E, Nukada T, Ueno T.Taylorella equigenitalis causes contagious equine metritis. Here we compared seven nucleic acid amplification tests for T. equigenitalis to select a rapid and reliable diagnostic method. The 95% detection limits of each assay varied greatly: real-time PCR had the lowest detection limit (0.77 fg/reaction); those of some of the conventional PCRs (cPCRs) were >100 fg/reaction. In experimentally infected samples, real-time PCR and semi-nested PCR showed the highest positive numbers (33 out of 42 samples), but two of the cPCRs detected only 2 and 7 positive results. Our results indicate that the us...
Amory H, Cesarini C, De Maré L, Loublier C, Moula N, Detilleux J, Saulmont M, Garigliany MM, Lecoq L.The objective of this retrospective study was to evaluate the clinical significance of fecal quantitative real-time polymerase chain reaction (qPCR) results when taking the cycle threshold values (Ct) into account. The study included 120 qPCR-positive fecal samples obtained from 88 hospitalized horses over a 2-year period. The mean Ct of the qPCR test was evaluated in regard to (1) clinical outcome and (2) systemic inflammatory response syndrome (SIRS) status (no SIRS, moderate SIRS, or severe SIRS) of the sampled horses. An ROC analysis was performed to establish the optimal cut-off Ct valu...
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...
Schwarzbach SV, Melo CF, Xavier PLP, Roballo KC, Cordeiro YG, Ambrósio CE, Fukumasu H, Carregaro AB.Osteoarthritis (OA) is one of the main locomotor disorders in horses. Although nonsteroidal anti-inflammatory drugs are the first-line treatment for OA, opioids could also be used. In previous studies, opioids showed promising anti-inflammatory and analgesic effects. In this study, we aimed to investigate the effects of two opioids (morphine and methadone) against inflammation in lipopolysaccharide (LPS)-stimulated synoviocytes by analyzing microsomal prostaglandin E synthase-1 (mPGES-1) and prostaglandin-endoperoxide synthase 2 (PTGS2) expression. Synoviocytes were obtained from the joints at...
Webb TL, Burnett RC, Avery AC, Olver CS.Characterization of anemia in horses presents a challenge, as they do not release reticulocytes into peripheral blood. Transferrin receptor (TfR) expression is highest on erythroid cells in people and rats, and measurement of a soluble serum form (sTfR) is used to quantify erythropoiesis in these species. We hypothesized that equine TfR (eTfR) expression is similar in quantity and distribution to that in these other species and thus has potential for characterization of the regenerative response in anemic horses. Objective: This study was conducted to clone and sequence the eTfR gene and measu...
Shchit IY, Ignatov KB, Biketov SF.Results of detection of Burkholderia mallei and Burkholderia pseudomallei DNA strains by LAMP (Loop-mediated Isothermal Amplification) and Real Time PCR are shown. It has been revealed that, in Real Time PCR, primers steadily detected DNA of those microorganism for the sequences of which they were designed. The above mentioned primers did not detect DNA of heterologous strains. During LAMP method no set of primers showed high analytical sensitivity and specificity. Primers did not detected DNA of all the strains under research to target genes of which they were not intended, but they were capa...
Hughes SB, Quan M, Guthrie A, Schulman M.The insulin-like growth factor system (insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor 1 receptor, insulin-like growth factor 2 receptor and six insulin-like growth factor-binding proteins) and insulin are essential to muscle metabolism and most aspects of male and female reproduction. Insulin-like growth factor and insulin play important roles in the regulation of cell growth, differentiation and the maintenance of cell differentiation in mammals. In order to better understand the local factors that regulate equine physiology, such as muscle metabolism a...
Sayasith K, Bouchard N, Boerboom D, Brown KA, Doré M, Sirois J.Ovulation is accompanied by a marked infiltration of leukocytes into thecal layers after the gonadotropin surge. P-selectin is known to play a critical role in the initial steps of leukocyte recruitment from the bloodstream during inflammation. Thus, the objective was to investigate the potential regulation of P-selectin by gonadotropins in equine preovulatory follicles. The full-length equine P-selectin cDNA was cloned by a combination of reverse transcription-polymerase chain reaction (RT-PCR) and 5'- and 3'-rapid amplification of cDNA ends. Results showed that equine P-selectin cDNA encodes...
Inflammatory myopathy and perivasculitis have been recently described in horses with chronic equine piroplasmosis (EP). These alterations may be linked to poor performances. The aims of this study were to evaluate the prevalence for EP in clinically healthy Italian Standardbred (IS) racehorses and to compare laboratory parameters and performance metrics between positive and negative horses. Real-time PCR was applied for the detection of T. equi and B. caballi positivity. Haematology parameters, blood chemistry results, subjective muscle mass scores, and performance metrics were compared betwee...
de Oliveira UV, Varjão JL, de Jesus Deiró AG, Maciel BM, Silva FL, Pinheiro AM, Gondim LFP, Munhoz AD.The aim of this study was to isolate from equids destined for slaughter in a Brazilian slaughterhouse. A total of 354 equids were analyzed, with blood samples collected from all the animals and samples of masseter muscle and brain tissue collected from 319 animals. A serological test was conducted to detect equids with specific antibodies for . Molecular detection of by nested PCR was performed on the tissue samples collected. Tissue samples were tested by murine bioassay in an attempt to isolate either the parasite or the parasite DNA. Real-time PCR was performed on the brain samples from 1...
de Freitas MG, Dos Santos GH, Silva DLZ, Costa TD, Borges DGL, Reckziegel GH, Cleveland H, do Nascimento Ramos CA, Pereira FB, de Almeida Borges F.The strategic control of equine gastrointestinal nematodes is based on epidemiology. The aim is the reduction of the population of parasites as a whole in the hosts and environment while seeking the number of treatments that achieves the balance between not risking animal health and not exerting selection pressure for resistant parasites. This is the first study in a tropical region to assess a strategic control protocol for equines. The study was conducted in the municipality of Anastácio in the state of Mata Gross do Sul, Brazil, over a two-year period from April 2021 to March 2023, involvi...
There are four genotypes of ovine papillomaviruses (OaPVs): OaPV1, OaPV2, and OaPV4, which are ovine delta papillomaviruses responsible for epithelial and mesenchymal cell infections, and OaPV3, an epitheliotropic Dyokappapapillomavirus associated with cutaneous tumors in sheep, including squamous cell carcinoma. Vaginal swabs of healthy mares were evaluated for the presence of PVs to investigate whether the vaginal virobiota of asymptomatic mares harbored OaPVs. High-performance digital droplet polymerase chain reaction (ddPCR) was used to quantitatively detect OaPV types 1-4 DNA in 94 vagina...
Cullinane A, Nelly M, Dayot L, Lukaseviciute G, Garvey M, Healy J, Gallagher R.This study describes a comparison of the detection of rotavirus in clinical samples from foals using two commercially available rapid antigen detection (RAD) kits, with the detection of rotavirus nucleic acid via a laboratory-based, in-house, real-time reverse transcription polymerase chain reaction (RT-PCR) assay. One hundred and forty freeze-thawed samples (70 that were RT-PCR-positive and 70 that were RT-PCR-negative on original tests) submitted to the diagnostic laboratory over a seven-year period were tested in addition to 123 fresh samples (15 RT-PCR-positive and 108 RT-PCR-negative) sub...
Bhoora RV, Mbaba TV, Troskie M, Ackermann RE, Collins NE.Theileria haneyi is an apicomplexan parasite closely related to Theileria equi, a known causative agent of equine piroplasmosis. The molecular distinction between these parasites relies on a nested polymerase chain reaction (PCR) assay, which has been reported to be unreliable. A recently reported indirect ELISA based on equi merozoite antigen 11 (Thema-11) of T. haneyi can detect geographically diverse T. haneyi strains. Since the ema-11 gene is exclusive to T. haneyi, it was chosen as the target for developing a TaqMan minor groove binder (MGB™) quantitative real-time PCR (qPCR). Published...
Santiviparat S, Suthithanakom S, Bhanpattanakul S, Srisuwattanasagul S, Melde K, Stout TAE, Tharasanit T.Advances in endometrial tissue engineering have enabled the combination of modified scaffolding materials with modern cell culture technologies. Genipin and three-dimensional (3D) printing have advanced cell-tissue engineering by enabling the precise layering of cell-containing matrices while ensuring low cytotoxicity. This study aimed to advance equine endometrial tissue engineering by designing customized collagen scaffolds using 3D printing technology, while optimizing the genipin concentration to avoid toxicity. Genipin was tested at concentrations of 4, 2, 1, 0.5, 0.25, 0.125, and 0 mM on...
Iduu NV, Raiford D, Cohen ND, Landrock KK, Wang C.We developed a fluorescence resonance energy transfer (FRET)-based real-time PCR (qPCR) assay coupled with high-resolution melting (HRM) analysis to differentiate subsp. (SEE) from subsp. (SEZ), two closely related equine pathogens with distinct clinical implications. The assay targets a region of the 23S rRNA gene containing single-nucleotide polymorphisms that differentiate the subspecies. By combining subspecies-specific probes with melting temperature () analysis, the assay accurately distinguished SEE, SEZ, and mixed infections without the need for post-PCR processing. The entire work...
Pusterla N, Lawton K, Barnum S, Vitomirov A, Anaya S, Naranatt P, Swadia H, Mendonsa E.The objective of this study was to evaluate a point-of-care (POC) PCR assay for the detection of Salmonella spp. in pooled environmental samples collected at an equine veterinary hospital. A total of 945 environmental samples were collected from high-risk areas, including ICU and isolation stalls, high-traffic areas, treatment rooms, and surgical suites. The environmental samples were collected using drag swabs placed in selenite broth and individually incubated at 35 °C for 20 h. Following the incubation period, 1 mL of up to 10 individual environmental samples were pooled together. Each poo...
Zhou Y, Yao X, Meng J, Wang J, Zeng Y, Li L, Ren W.The Kazakh horse is a highly valuable indigenous Chinese breed known for its use in both milk and meat production. However, the mechanisms underlying color variation in the abdominal adipose tissue of this breed remain poorly understood. In this study, the sequencing of non-coding RNAs (ncRNAs) was conducted on abdominal adipose tissue of different colors from Kazakh horses, with the aim of investigating the molecular mechanisms responsible for this variation. A total of 205 differentially expressed long non-coding RNAs (DELncRNAs) including , , and ; 52 differentially expressed microRNAs (DEm...
McCreight KA, Barbosa LN, Odoi A, Reed P, Rajeev S.We estimated the seroprevalence in dogs, cats, and horses from Tennessee, USA, using the microscopic agglutination test (MAT) against 12 serovars. We observed seropositivity in 110 of 374 (29.4%) dogs, 21 of 170 (12.4%) cats, and 42 of 88 (47.7%) horses. The highest seroprevalence was observed for serovars Autumnalis (74.6%) in dogs, and Bratislava in cats (42.9%) and horses (95.2%). We found a significant level of potential cross-reactivity between multiple serovars tested, with highest cross-reactivity to serovar Autumnalis in dogs. seroprevalence was significantly higher in vaccinated ...
Huang C, Wang J, Ruan Z, Wu J, Lin Y, Cao C, Yang J, Weng Q, Jin Y, Chen P, Hua Q.African horse sickness (AHS) is an acute infectious disease of equids caused by the AHS virus (AHSV), which can cause up to 90% mortality in naive horses. Reliable and rapid diagnosis is crucial for the surveillance and control of AHSV. As one of the AHSV detection methods recommended by World Organization for Animal Health (WOAH), the RT-qPCR assay has the drawbacks such as complex operation, expensive instruments, and long detecting time, which limit its application in simple laboratories or outdoors. In this study, a real-time reverse transcription multienzyme isothermal rapid amplification...
Balasuriya UB.The primary goals of this chapter are to discuss common viral RNA isolation and purification methods that are routinely used by various diagnostic laboratories, to highlight the advantages and drawbacks of each method, and to identify the most suitable and reliable method to increase the sensitivity and specificity of RT-PCR assays for the detection of equine influenza virus (EIV) in clinical specimens. Our experiences and review of literature show that magnetic bead-based nucleic extraction methods (manual and automatic) work well for isolation and purification of EIV RNA from nasal swab spec...
Szczerba-Turek A, Bancerz-Kisiel A, Lipczyńska K, Siemionek J, Raś A, Platt-Samoraj A, Szweda W.The aim of the study was to evaluate a novel diagnostic scheme which combines quantitative PCR and High-Resolution Melting (qPCR-HRM) curve analysis for rapid differentiation based on E5 partial CDS of bovine papillomavirus type 1 or 2 (BPV-1 or BPV-2), and to perform a phylogenetic analysis of the complete CDS of the E5 gene of BPV detected in equine sarcoids. Samples of 38 skin lesions obtained from 27 horses were collected for molecular examinations. All lesions were clinically diagnosed as sarcoids, but results of histopathological examinations did not always corroborate the clinical diagn...
Balasuriya UBR.The primary goals of this chapter are to discuss common viral RNA isolation and purification methods that are routinely used by various diagnostic laboratories and to highlight the advantages and drawbacks of each method and to identify the most suitable and reliable method to increase the sensitivity and specificity of RT-PCR assays for the detection of equine influenza virus (EIV) in clinical specimens. Our experiences and review of literature show that magnetic bead-based nucleic extraction methods (manual and automatic) work well for isolation and purification of EIV RNA from nasal swab sp...
