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.
Chen K, Hu Z, Yang G, Guo W, Qi T, Liu D, Wang Y, Du C, Wang X.Equine Piroplasmosis (EP) is a tick-borne disease caused by three apicomplexan protozoan parasites, Theileria equi (T. equi), Babesia caballi (B. caballi) and T. haneyi, which can cause similar clinical symptoms. There are five known 18S rRNA genotypes of T. equi group (including T. haneyi) and three of B. caballi. Real-time PCR methods for detecting EP based on 18S rRNA analysis have been developed, but these methods cannot detect all genotypes of EP in China, especially genotype A of T. equi. In this study, a duplex real-time PCR detection method was developed for the simultaneous detection ...
Rocafort-Ferrer G, Leblond A, Joulié A, René-Martellet M, Sandoz A, Poux V, Pradier S, Barry S, Vial L, Legrand L.Equine piroplasmosis (EP) is a tick-borne disease caused by Babesia caballi and Theileria equi that is potentially emerging in non-endemic countries. We conducted a descriptive study to investigate EP prevalence and spatial distribution in an endemic region: the Camargue and the Plain of La Crau in France. In spring 2015 and 2016, we carried out sampling at stables (total n = 46) with a history of horses presenting chronic fever or weight loss. Overall, we collected blood from 632 horses, which were also inspected for ticks; these horses had been housed in the target stables for at least 1...
Rizk MA, El-Sayed SAE, Eltaysh R, Igarashi I.Equine piroplasmosis is a tick-transmitted disease that is considered one of the most serious infectious diseases affecting equines. Searching for novel antipiroplasm drugs remains indispensable due to the emergence and spreading of resistant piroplasm parasites against the limited currently used drugs, diminazene aceturate and imidocarb dipropionate. Therefore, novel drugs with specified targets need to be identified and exploited. The inhibitory effects of Medicines for Malaria Venture (MMV) Malaria Box compounds with potent in vitro anti-equine piroplasmosis activity were evaluated against ...
Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Nagata SI.One method of gene doping in horseracing is administering of exogenous genetic materials, known as transgenes. Several polymerase chain reaction (PCR)-based methods have been developed for detecting transgenes with high sensitivity and specificity. However, novel designs for reference materials (RMs) and/or positive template controls (PTCs) are necessary for simultaneous analysis of multiple transgene targets. In this study, we designed and developed a novel RM for simultaneously detecting multiple targets via microfluidic quantitative PCR (MFQPCR). Twelve equine genes were selected as targets...
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...
Page AE, Wood C, Partridge E, Horohov DW, Adam E.The use of lipopolysaccharide to induce a localized source of inflammation (acute synovitis) and allow for monitoring of changes in systemic mRNA expression has been recently reported. Here, the goal was to maintain a significant systemic mRNA response while limiting the severity of lameness such that this model can be used to examine the effects of various anti-inflammatory treatment modalities on mRNA expression. Three mixed breeds, four-year-old geldings were utilized for this study. One milliliter of phosphate-buffered saline containing 1,000 ng or less of lipopolysaccharide from E. coli O...
Flanagan S, Rowe Á, Duggan V, Markle E, O'Brien M, Barry G.Warmblood Fragile Foal syndrome (WFFS) is an autosomal recessive condition that affects the maturation of collagen in affected foals. Foals affected with the disease typically die or are euthanised shortly after birth. WFFS is caused by a single nucleotide change at position 2032 of the equine PLOD1 gene, causing an impairment of the wild-type enzyme. A commercial test for the causative genetic mutation is currently available from companies operating under licence from Cornell University but it has limitations. This test requires amplification of a region of the PLOD1 gene encompassing the sit...
Amaral A, Fernandes C, Szóstek-Mioduchowska A, Lukasik K, Rebordão MR, Pinto-Bravo P, Skarzynski DJ, Ferreira-Dias G.Cathepsin G (CAT) is a protease released by neutrophils when forming neutrophil extracellular traps that was already associated with inducing type I collagen (COL1) in equine endometrium in vitro. Endometrosis is a fibrotic condition mainly characterized by COL1 deposition in the equine endometrium. The objective was to evaluate if noscapine (an alkaloid for cough treatment with anti-neoplastic and anti-fibrotic properties) would reduce transcription (evaluated by qPCR) and COL1 protein relative abundance (evaluated by western blot) induced by CAT in equine endometrial explants from follicula...
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...
McGregor BL, Rozo-Lopez P, Davis TM, Drolet BS.Vesicular stomatitis (VS) is a reportable viral disease which affects horses, cattle, and pigs in the Americas. Outbreaks of vesicular stomatitis virus New Jersey serotype (VSV-NJ) in the United States typically occur on a 5-10-year cycle, usually affecting western and southwestern states. In 2019-2020, an outbreak of VSV Indiana serotype (VSV-IN) extended eastward into the states of Kansas and Missouri for the first time in several decades, leading to 101 confirmed premises in Kansas and 37 confirmed premises in Missouri. In order to investigate which vector species contributed to the outbrea...
Alnaeem A, Shawaf T, Ali AM, Hemida MG.In the current study, we are investigating the viral causes of some respiratory clinical signs in some animals belongs to the family Equidae in eastern Saudi Arabia (ESA) during winter- 2019. We observed the progression of severe respiratory clinical signs among some horses, donkeys, and ponies in the ESA. Animals showed rapid respiration, fever, nasal discharges (started as serous then changed into mucopurulent with the progression of the infection per some animals). We conducted a longitudinal study to monitor the progression of this outbreak. We conducted molecular surveillance for the infl...
Mok CH, MacLeod JN.Within developing synovial joints, interzone and anlagen cells progress through divergent chondrogenic pathways to generate stable articular cartilage and transient hypertrophic anlagen cartilage, respectively. Understanding the comparative cell biology between interzone and anlagen cells may provide novel insights into emergent cell-based therapies to support articular cartilage regeneration. The aim of this study was to assess the kinetics of gene expression profiles in these skeletal cell lines after inducing chondrogenesis in culture. Interzone and anlagen cells from seven equine fetuses w...
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...
Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Nagata SI.Gene doping is a threat to fair competition in sports, both human and equestrian. One method of gene doping is to administer exogenous genetic materials, called transgenes, into the bodies of postnatal humans and horses. Polymerase chain reaction (PCR)-based transgene detection methods such as digital PCR and real-time PCR have been developed for gene doping testing in humans and horses. However, the significance of PCR inhibitors in gene doping testing has not been well evaluated. In this study, we evaluated the effects of PCR inhibitors on transgene detection using digital PCR and real-time ...
Kim SH, Lee JS, Lee JH, Kim YJ, Choi JG, Lee SK, Kim HJ, Yang SJ, Park T, Lee SK, Kang HE, Jeoung HY, Park JY.Venereal diseases caused by bacteria are important to the equine industry due to economic losses caused by decline of conception rate in breeding horses. Therefore, identification of infected animals as well as the implementation of appropriate managerial procedures based on accurate diagnosis is critical. In this study, two types of multiplex real-time polymerase chain reaction with high sensitivity and specificity were developed for the simultaneous detection and differentiation of five commonly associated bacterial pathogens of venereal diseases in horses, consisting of Taylorella equigenit...
Bradbery AN, Coverdale JA, Hammer CJ, Dunlap KA, Leatherwood JL, Satterfield MC.Skeletal muscle plays an integral role in the ability of a horse to perform at high levels. Shifts in skeletal muscle development in response to maternal plane of nutrition may have substantial and lasting impacts on athletic performance and whole-body metabolism. Therefore, sixteen Quarter Horse mares were used in a completely randomized design and maintained at a body condition score (BCS) 6 until start of third trimester. On d 235 of gestation, mares were randomly assigned to receive one of two dietary treatments with a diet formulated to meet requirements during late gestation (CON; n = ...
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 ...
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...
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 ...
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...
Equine piroplasmosis (EP) is a disease of equids caused by Theileria equi and Babesia caballi, members of the order Piroplasmida, transmitted by several species of ticks. As the disease is endemic in many countries, a clinical examination or a serological test are required prior to movement of horses to prove freedom from infection and to avoid the introduction of EP with its sanitary and economic impact, especially in areas where it is absent. Currently, numerous diagnostic PCR protocols are available, some of which are recommended by the World Organisation for Animal Health (OIE). In order t...
Pinto-Bravo P, Rebordão MR, Amaral A, Fernandes C, Galvão A, Silva E, Pessa-Santos P, Alexandre-Pires G, Roberto da Costa RP, Skarzynski DJ....The oviduct presents the ideal conditions for fertilization and early embryonic development. In this study, (i) vascularization pattern; (ii) microvascular density; (iii) transcripts of angiogenic factors (, , ) and their receptors-, , , respectively, and (iv) the relative protein abundance of those receptors were assessed in cyclic mares' oviducts. The oviductal artery, arterioles and their ramifications, viewed by means of vascular injection-corrosion, differed in the infundibulum, ampulla and isthmus. The isthmus, immunostained with CD31, presented the largest vascular area and the highest ...
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...
Jaferi M, Mozaffari A, Jajarmi M, Imani M, Khalili M.There are few reports about Q fever in horse populations worldwide. This study aimed to detect the C. burnetii infection by serologic and molecular confirmation using commercial ELISA kit and real-time PCR in the East of Iran a region highly endemic. A total of 177 blood samples and 115 vaginal swabs were randomly collected from horses in East of Iran. The sera samples were analyzed for anti C.burnetii Ig G antibodies by a commercial ELISA kit and nucleic acid extraxted from vaginal samples were used to determine the C. burnetii DNA by real-time PCR assay. Antibodies were detected in 5.64 % (1...
Bernardino P, James K, Barnum S, Corbin R, Wademan C, Pusterla N.Equine rhinitis B virus (ERBV) has been given little attention by practitioners compared to other respiratory viruses, mainly because of the lack of diagnostic modalities and association with clinical disease. The objective of the study was to determine the frequency of detection of ERBV in nasal secretions from 6568 horses with acute onset of respiratory signs. ERBV-positive qPCR results from nasal secretions submitted to a molecular diagnostic laboratory from 2013 to 2019 were reviewed. A total of 333 ERBV qPCR-positive samples (5.1%) were detected with increasing yearly frequency since the ...
Lisowski ZM, Lefevre L, Mair TS, Clark EL, Hudson NPH, Hume DA, Pirie RS.Studies in rodents and humans have demonstrated that intestinal manipulation or surgical trauma initiates an inflammatory response in the intestine which results in leucocyte recruitment to the muscularis externa causing smooth muscle dysfunction. Objective: To examine the intestinal inflammatory response in horses undergoing colic surgery by measuring relative differential gene expression in intestinal tissues harvested from surgical colic cases and control horses. Methods: Prospective case-control study. Methods: Mucosa and muscularis externa were harvested from healthy margins of resected s...
Bernardino PN, Smith WA, Conrad PA, Packham AE, Tamez-Trevino E, Barnum S, Pusterla N.An ante-mortem diagnosis of equine protozoal myeloencephalitis (EPM) is presently based on clinical presentation, immunodiagnostics performed on serum and cerebrospinal fluid (CSF), and ruling out other neurological disorders. Molecular techniques introduce a novel and promising approach for the detection of protozoal agents in CSF. Hypothesizing that real-time PCR (rtPCR) can be a useful complement to EPM diagnostics, 210 CSF samples from horses suspected of neurological disease with EPM included as a differential diagnosis were tested using rtPCR to detect Sarcocystis neurona DNA and immunod...
Yang F, Richardson DW.The tendon is highly prone to injury, overuse, or age-related degeneration in both humans and horses. Natural healing of injured tendon is poor, and cell-based therapeutic treatment is still a significant clinical challenge. In this study, we extensively investigated the expression of tenogenic genes in equine bone marrow mesenchymal stem cells (BMSCs) and tenocyte-derived induced pluripotent stem cells (teno-iPSCs) stimulated by growth factors (TGF-3 and BMP12) combined with ectopic expression of tenogenic transcription factor MKX or cyclic uniaxial mechanical stretch. Western blotting reveal...
Kadivar A, Rashidzadeh H, Davoodian N, Nazari H, Dehghani Tafti R, Heidari Khoei H, Seidi Samani H, Modaresi J, Ahmadi E.Accurate diagnosis of foetal sex in pregnant mare is helpful for many breeders, both for private or commercial purposes. In this study, in order to pre-natal foetal sexing in equine, we used TaqMan duplex real-time PCR to detect the specific regions of SRY and TSPY genes on extracted cell-free foetal DNA from maternal blood. Peripheral blood samples from 50 pregnant Arabian mares with singleton foetuses were collected. Cell-free foetal DNA was extracted from maternal plasma, and duplex real-time PCR assays were performed with TaqMan probes and primers. Amplification of glyceraldehyde-3-phospha...
Sears WJ, Qvarnstrom Y, Dahlstrom E, Snook K, Kaluna L, Baláž V, Feckova B, Šlapeta J, Modry D, Jarvi S, Nutman TB.Angiostrongylus cantonensis (Ac), or the rat lungworm, is a major cause of eosinophilic meningitis. Humans are infected by ingesting the 3rd stage larvae from primary hosts, snails, and slugs, or paratenic hosts. The currently used molecular test is a qPCR assay targeting the ITS1 rDNA region (ITS1) of Ac. In silico design of a more sensitive qPCR assay was performed based on tandem repeats predicted to be the most abundant by the RepeatExplorer algorithm. Genomic DNA (gDNA) of Ac were used to determine the analytical sensitivity and specificity of the best primer/probe combination. This assay...
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...
Hughes KJ, Nicolson L, Da Costa N, Franklin SH, Allen KJ, Dunham SP.Inflammatory airway disease (IAD) is a common disorder of performance horses and is associated with poor performance and accumulation of mucus and inflammatory cells in lower airway secretions. Horses with IAD frequently have increased relative counts of neutrophils in bronchoalveolar lavage fluid (BALF); less commonly relative counts of eosinophils and/or mast cells may be increased. The aetiopathogenesis of IAD is unknown and may involve innate and/or acquired immune responses to various factors including respirable dust constituents, micro-organisms, noxious gases and unconditioned air. The...
Balaskas P, Green JA, Haqqi TM, Dyer P, Kharaz YA, Fang Y, Liu X, Welting TJM, Peffers MJ.Ageing is a leading risk factor predisposing cartilage to osteoarthritis. However, little research has been conducted on the effect of ageing on the expression of small non-coding RNAs (sncRNAs). RNA from young and old chondrocytes from macroscopically normal equine metacarpophalangeal joints was extracted and subjected to small RNA sequencing (RNA-seq). Differential expression analysis was performed in R using package DESeq2. For transfer RNA (tRNA) fragment analysis, tRNA reads were aligned to horse tRNA sequences using Bowtie2 version 2.2.5. Selected microRNA (miRNAs or miRs) and small nucl...
Quinlivan M, Dempsey E, Ryan F, Arkins S, Cullinane A.Equine influenza is a cause of epizootic respiratory disease of the equine. The detection of equine influenza virus using real-time Light Cycler reverse transcription (RT)-PCR technology was evaluated over two influenza seasons with the analysis of 171 samples submitted for viral respiratory disease. Increased sensitivity was found in overall viral detection with this system compared to Directigen Flu A and virus isolation, which were 40% and 23%, respectively, that of the RT-PCR. The assay was also evaluated as a viable replacement for the more traditional methods of quantifying equine influe...
Laan TT, Bull S, Pirie R, Fink-Gremmels J.When challenged with allergens and pro-inflammatory agents, such as Aspergillus fumigatus (AF), hay dust solution (HDS) and lipopolysaccharide (LPS), the innate immune response will not only activate the immune system but also increase the amount of pro-inflammatory cytokines in the bronchoalveolar space. The aim of this study was to assess the response of equine alveolar macrophages to different aerosolized challenges and to investigate the differences in this response between horses susceptible or nonsusceptible to recurrent airway obstruction (RAO). Seven susceptible and 5 nonsusceptible ho...
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...
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...
Almeida J, Ball BA, Conley AJ, Place NJ, Liu IK, Scholtz EL, Mathewson L, Stanley SD, Moeller BC.Anti-Müllerian hormone (AMH), a member of the transforming growth factor β superfamily of growth and differentiation factors, is expressed in granulosa cells of preantral and small antral ovarian follicles. In humans, AMH appeared to regulate recruitment and growth of small ovarian follicles. Furthermore, circulating AMH concentrations were elevated in women with granulosa-cell tumors (GCT). In the horse, GCTs are the most common tumor of the ovary, and a variety of endocrine assays have been used to diagnose presumptive GCTs. The objectives of the present study were to validate a heterologo...
Peat FJ, Colbath AC, Bentsen LM, Goodrich LR, King MR.This study aimed to examine the influence of neodymium-doped yttrium aluminum garnet (Nd:YAG) laser irradiation on equine bone marrow-derived mesenchymal stem cell (MSC) viability, proliferation, and cytokine expression in vitro. Background: Photobiomodulation of cells using monochromatic light is a technique designed to influence cellular processes. Previous studies have shown dose-dependent effects of low-level laser irradiation on cell proliferation and cytokine expression in a range of cell types and species. Evidence for the influence of 1064 nm wavelength near-infrared irradiation on M...
Stasiak K, Dunowska M, Rola J.Equid herpesviruses (EHVs) are widespread in equine populations worldwide. While the infection with equine α-herpesviruses (EHV-1 and EHV-4) has been linked to several clinical outcomes, the pathogenic potential for equine γ-herpesviruses (EHV-2 and EHV-5) is still unclear. The objective of the current study was to determine the prevalence of infection with EHVs among Polish horses, to investigate factors associated with EHV infections among horses sampled, and to determine genetic variability within Polish EHV-2 isolates. Virus-specific real-time PCR assays were used for detection of EHV-1,...
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 ...
Remacha AR, Barrachina L, Álvarez-Arguedas S, Ranera B, Romero A, Vázquez FJ, Zaragoza P, Yañez R, Martín-Burriel I, Rodellar C.The immunomodulatory capacities of mesenchymal stem cells (MSCs) have made them the subject of increased clinical interest for tissue regeneration and repair. We have studied the immunomodulatory capacity of equine MSCs derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) in cocultures with allogeneic peripheral blood mononuclear cells (PBMCs). Different isoforms and concentrations of phytohaemaglutinin (PHA) were tested to determine the best stimulation conditions for PBMC proliferation and a proliferation assay was performed for 7 days to determine the optimal day of stimulation o...
McGregor BL, Rozo-Lopez P, Davis TM, Drolet BS.Vesicular stomatitis (VS) is a reportable viral disease which affects horses, cattle, and pigs in the Americas. Outbreaks of vesicular stomatitis virus New Jersey serotype (VSV-NJ) in the United States typically occur on a 5-10-year cycle, usually affecting western and southwestern states. In 2019-2020, an outbreak of VSV Indiana serotype (VSV-IN) extended eastward into the states of Kansas and Missouri for the first time in several decades, leading to 101 confirmed premises in Kansas and 37 confirmed premises in Missouri. In order to investigate which vector species contributed to the outbrea...
Lei R, Wang X, Zhang D, Liu Y, Chen Q, Jiang N.Equine piroplasmosis (EP) is a severe disease of horses caused by the tick-borne protozoa Theileria equi (T. equi) and Babesia caballi (B. caballi). Infectious carriers are not always symptomatic, meaning there is a risk to non-enzootic areas. Regulatory tests for EP include sero-epidemiological methods for equine babesiosis, but these lack specificity due to cross-reactivity with other Babesia species. In this study, we present a real-time quantitative recombinase polymerase amplification (qRPA) method for fast simultaneous detection of both T. equi and B. caballi. In this method, primers and...
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...
Capomaccio S, Cappelli K, Barrey E, Felicetti M, Silvestrelli M, Verini-Supplizi A.It is known that moderate physical activity may have beneficial effects on health, whereas strenuous effort induces a state resembling inflammation. The molecular mechanisms underlying the cellular response to exercise remain unclear, although it is clear that the immune system plays a key role. It has been hypothesized that the physio-pathological condition that develops in athletes subjected to heavy training is caused by derangement of cellular immune regulation. The purpose of the present study was to obtain information on endurance horse gene transcription under strenuous conditions and t...
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...
Murphy BA, Wagner AL, McGlynn OF, Kharazyan F, Browne JA, Elliott JA.Circadian rhythms are endogenously generated 24-h oscillations that coordinate numerous aspects of mammalian physiology, metabolism and behaviour. The existence of a molecular circadian clock in equine skeletal muscle has previously been demonstrated. This study investigates how the circadian 24-h expression of exercise-relevant genes in skeletal muscle is influenced by a regular exercise regime. Mid-gluteal, percutaneous muscle biopsies were obtained over a 24-h period from six Thoroughbred mares before and after an 8-week exercise programme. Real-time qPCR assays were used to assess the expr...
Bogaert L, Martens A, Kast WM, Van Marck E, De Cock H.Bovine papillomavirus (BPV)-1 and -2 is linked to equine sarcoids, a commonly observed skin tumor in horses that is of considerable veterinary importance. Previous studies using in situ hybridization have detected BPV DNA only in fibroblasts and not in keratinocytes of sarcoids. In contrast, normal equine skin latently infected with BPV shows a dysplastic epithelium without dermal changes, similar to lesions induced by other papillomavirus types infecting the epithelium. The first goal of our study was to describe the epidermal and dermal characteristics of several stages in sarcoid developmen...
Cavalleri JV, Korbacska-Kutasi O, Leblond A, Paillot R, Pusterla N, Steinmann E, Tomlinson J.Horses and other equids can be infected with several viruses of the family Flaviviridae, belonging to the genus Flavivirus and Hepacivirus. This consensus statement focuses on viruses with known occurrence in Europe, with the objective to summarize the current literature and formulate clinically relevant evidence-based recommendations regarding clinical disease, diagnosis, treatment, and prevention. The viruses circulating in Europe include West Nile virus, tick-borne encephalitis virus, Usutu virus, Louping ill virus and the equine hepacivirus. West Nile virus and Usutu virus are mosquito-bor...
Pusterla N, Sandler-Burtness E, Barnum S, Hill LA, Mendonsa E, Khan R, Portener D, Ridland H, Schumacher S.The objective of this study was to determine detection frequency of respiratory viruses (equine influenza virus [EIV], equine herpesvirus-1 [EHV-1], EHV-2, EHV-4, EHV-5, equine rhinitis A virus [ERAV], ERBV) and bacteria (Streptococcus equi ss. equi[S. equi], S. equi ss. zooepidemicus[S. zooepidemicus]) in 162 nasal secretions and 149 stall swabs from healthy sport horses attending a spring show in California. Nasal and stall swabs were collected at a single time point and analyzed using qPCR. The detection frequency of respiratory pathogens in nasal secretions was 38.9% for EHV-2, 36.4% for E...
Castagnetti C, Mariella J, Pirrone A, Cinotti S, Mari G, Peli A.To evaluate and compare the gene expression of interleukin(IL)-1β, IL-8, and interferon-γ during the first 72 hours after birth in healthy foals and during the first 72 hours after hospitalization in sick neonatal foals and investigate correlations of clinicopathologic variables with cytokine expressions in healthy and sick neonatal foals. Methods: 33 foals < 7 days old (10 healthy foals, 7 foals with sepsis, 6 foals with peripartum asphyxia syndrome, and 12 foals with other diseases [2 with failure of passive transfer of immunity only were not further evaluated]). Methods: A blood sample...
D'Ascola A, Bruschetta G, Zanghì G, Campo S, Medica P, Campana S, Ferlazzo G, Gibbs BF, Ferlazzo AM.Serotonin (5-HT) is a neurohormone transported from plasma into platelets and leukocytes by a specific transporter (SERT). While it is known that the brain 5-HT system is modulated by physical exercise, the peripheral serotoninergic response to exercise is not yet fully elucidated. In particular, this study aimed to evaluate changes in plasma 5-HT levels and equine leukocyte SERT expression in response to treadmill exercise in untrained horses. Analyses were carried out pre- and post-treadmill exercise. 5-HT plasma levels were analysed by HPLC. Leukocytes and platelets were isolated to perform...
Frellstedt L, Waldschmidt I, Gosset P, Desmet C, Pirottin D, Bureau F, Farnir F, Franck T, Dupuis-Tricaud MC, Lekeux P, Art T.In humans, strenuous exercise causes increased susceptibility to respiratory infections associated with down-regulated expression of Toll-like receptors (TLRs) and costimulatory and antigen-presenting molecules. Lower airway diseases are also a common problem in sport and racing horses. Because innate immunity plays an essential role in lung defense mechanisms, we assessed the effect of acute exercise and training on innate immune responses in two different compartments. Blood monocytes and pulmonary alveolar macrophages (PAMs) were collected from horses in untrained, moderately trained, inten...
Perkins GA, Goodman LB, Dubovi EJ, Kim SG, Osterrieder N.Early identification of inhalation-transmitted equine herpesvirus type 1 (EHV-1) infections has been facilitated by the availability of a number of real-time quantitative PCR (qPCR) tests. A direct comparison between nasal swab qPCR and traditional virus isolation (VI) requires a method for normalizing the qPCR samples and controlling for PCR inhibitors present in some clinical samples. Objective: To quantify EHV-1 shedding in viral swabs using an internal control and to compare fast qPCR to VI for the detection of EHV-1 in nasal swabs from horses. Methods: Fifteen horses experimentally infect...
Singh Suri S, Janardhan KS, Parbhakar O, Caldwell S, Appleyard G, Singh B.Toll-like receptor (TLR) is a key component in launching innate immune response to microbial challenge. TLR4 and TLR2 are recognized as specific receptors for components of Gram-negative and Gram-positive bacteria, respectively. Horses are extremely sensitive to endotoxin-induced cardiopulmonary distress and mortality which causes significant economic losses. To date, there are no data on the expression of TLR4 and TLR2 in horse lungs. Therefore, we examined the expression of TLR4 and TLR2 in lungs from normal or Escherichia coli lipopolysaccharide (E. coli LPS; 50 ng/kg; iv) treated horses. W...
Mapes S, Rhodes DM, Wilson WD, Leutenegger CM, Pusterla N.Fifty-five isolates of Escherichia coli from septicaemic neonatal foals were used to validate five real-time pcr assays targeting different known virulence factor genes: curli fibre (csgD), ferric hydroxamate uptake (fhuA), type 1A pilin (fimA), aerobactin (lutA) and yersiniabactin (fyuA). A pcr assay targeting a universal sequence of the bacterial 16S rrna gene served as quality control. The pcr assays showed good analytical specificity and sensitivity on the basis of sequencing the pcr products, their lack of cross-reactivity with non-E coli organisms, high amplification efficiency and a lim...
Zhang YW, Davis EG, Bai J.Quantitative reverse transcription polymerase chain reaction (RT-PCR) has become a basic, reliable and sensitive modern technique, in both biological research and clinical diagnosis, for investigation of gene expression and validation of cDNA microarray analysis. Accurate mRNA quantification using quantitative RT-PCR commonly requires data normalization through stable housekeeping genes (HKGs). Selection of HKGs for data normalization is critical for accurate mRNA quantification. Our objective was to evaluate a set of candidate HKGs as internal controls for gene expression studies using quanti...
Quan M, Lourens CW, MacLachlan NJ, Gardner IA, Guthrie AJ.Nucleotide sequences of 52 South African isolates of African horse sickness virus (AHSV) collected during 2004-2005 and including viruses of all nine AHSV serotypes, were used to design and develop a duplex real-time reverse transcription quantitative PCR (RT-PCR) assay targeting the VP7 (S8) and NS2 (S9) genes of AHSV. The assay was optimized for detection of AHSV in fresh and frozen blood of naturally infected horses. Assay performance was enhanced using random hexamers rather than gene-specific primers for RT, and with denaturation of double-stranded RNA in the presence of random hexamers. ...
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...
