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.
Lu Z, Chambers TM, Boliar S, Branscum AJ, Sturgill TL, Timoney PJ, Reedy SE, Tudor LR, Dubovi EJ, Vickers ML, Sells S, Balasuriya UB.The objective of this study was to develop and evaluate new TaqMan real-time reverse transcription-PCR (rRT-PCR) assays by the use of the minor groove binding probe to detect a wide range of equine influenza virus (EIV) strains comprising both subtypes of the virus (H3N8 and H7N7). A total of eight rRT-PCR assays were developed, targeting the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of the two EIV subtypes. None of the eight assays cross-reacted with any of the other known equine respiratory viruses. Three rRT-PCR assays (EqFlu NP, M, and HA3) which can detect strains of th...
Reyner CL, Wagner B, Young JC, Ainsworth DM.To examine gene expression of selected cytokines in pulmonary mononuclear cells isolated from healthy horses and horses susceptible to recurrent airway obstruction (RAO), and to determine whether interleukin (IL)-17 and IL-23 were associated with pulmonary inflammation. Methods: 6 RAO-susceptible and 5 healthy horses. Methods: Bronchoalveolar lavage cells were retrieved from horses that were stabled and fed dusty hay for 24 hours. Lavage cells devoid of neutrophils were incubated for 24 hours with solutions of PBS, hay dust, lipopolysaccharide, or B-glucan. Gene expression of IL-17, IL-23 (p19...
Pusterla N, Byrne BA, Hodzic E, Mapes S, Jang SS, Magdesian KG.A quantitative real-time (RT)-PCR assay was developed to detect Salmonella spp. in the feces of 911 equine species admitted to a veterinary hospital. Fresh feces and feces enriched for 24h in selenite broth were assessed by conventional culture and by RT-PCR targeting the Salmonella invA gene. The detection limit for the RT-PCR assay was 3 and 10 organisms, respectively, when spiked samples were purified from selenite broth and feces. The analytical specificity was 100% based on the detection of a panel of 40 salmonella serotypes from five serogroups and the lack of cross-reactivity with non-r...
Peretich AL, Abbott LL, Andrews FM, Dhar MS.To determine whether expression of mRNA for sodium-potassium adenosine-triphosphatase (NAKA) and sodium-hydrogen exchanger (NHE) in samples of the nonglandular portion of the equine gastric mucosa was altered by exposure to volatile fatty acids (VFAs) in an acidic environment. Methods: 10 horses (5 or = 12 years old). Methods: Samples of the nonglandular portion of the gastric mucosa were collected and exposed in Ussing chambers to Ringer's solution (control samples), Ringer's solution containing a mixture of VFAs (pH, 1.5 or 4.0), or Ringer's solution containing acetic acid (pH, 1.5 or 4.0)....
Pusterla N, Jackson R, Mapes SM, Noland J, Stenbom RM, Gebhart C.The humoral immune response and fecal shedding of Lawsonia intracellularis was investigated in 20 weanling foals following intra-rectal administration of frozen-thawed or lyophilized avirulent live L. intracellularis vaccine. Foals received either 30 mL frozen-thawed or lyophilized vaccine intra-rectally, given twice, 4 weeks apart. Serum samples from each foal were collected every 4 weeks for 16 weeks following the first vaccination and tested for anti-L. intracellularis specific IgG by immunoperoxidase monolayer assay. Rectal swabs were collected every other day following the first vaccinati...
Echigoya Y, Sato T, Itou T, Endo H, Sakai T.The species-specific properties of LDH isozymes are essentially determined by M (muscle) and H (heart) subunit proteins encoded by the LDHA and LDHB genes, respectively. In the present study, we molecularly characterized the full-length equine lactate dehydrogenase A (eLDHA) and B (eLDHB) cDNAs. The eLDHA cDNA consisted of a 999-bp open reading frame (ORF), while the eLDHB and newly acquired bat LDHB consisted of a 1002-bp ORF, which is 3 bp shorter than the LDHB ORF of other registered mammals. The alignment of amino acid sequences showed that eLDHA acquired positively charged His 88 and 226,...
van den Burg MM, Eizema K, de Graaf-Roelfsema E, van Breda E, Wijnberg ID, van der Kolk JH, Everts ME.To investigate the effects of acute exercise and long-term training on Na(+),K(+)-ATPase content, mRNA isoforms, and protein concentration in equine muscle. Methods: 6 Standardbreds. Methods: Horses performed a bout of exercise on a treadmill before and after 18 weeks of combined interval and endurance training. Muscle biopsy specimens were obtained from vastus lateralis muscle (VLM) and pectoralis descendens muscle (PDM) before and after exercise. The Na(+),K(+)-ATPase content, mRNA isoforms, and protein concentrations were determined by use of [(3)H]ouabain binding, real-time PCR assay, and ...
Bosch G, de Mos M, van Binsbergen R, van Schie HT, van de Lest CH, van Weeren PR.Extracorporeal shock wave therapy (ESWT) is frequently used in equine practice, but little is known about its biological action. Objective: To study the effects of ESWT on matrix structure and gene expression levels in normal, physiologically loaded tendinous structures in ponies. Methods: Six Shetland ponies, free of lameness and with ultrasonographically normal flexor and extensor tendons and suspensory ligaments (SL), were used. ESWT was applied at the origin of the suspensory ligament and the mid-metacarpal region of the superficial digital flexor tendon (SDFT) 6 weeks prior to sample taki...
Faleiros RR, Leise BB, Westerman T, Yin C, Nuovo GJ, Belknap JK.C-X-C motif ligand 1 (CXCL1) is an important chemokine of epithelial origin in rodents and humans. Objective: To assess in vivo and in vitro the regulation of CXCL1 in equine laminitis. Methods: Twenty adult horses. Methods: Real-time quantitative polymerase chain reaction (PCR) was used to assess expression of CXCL1 in samples of laminae, liver, skin, and lung from the black walnut extract (BWE) model of laminitis, and in cultured equine epithelial cells (EpCs). Tissue was obtained from control animals (CON, n = 5), and at 1.5 hours (early time point [ETP] group, n = 5), at the onset of leuko...
Perkins GA, Goodman LB, Tsujimura K, Van de Walle GR, Kim SG, Dubovi EJ, Osterrieder N.A single nucleotide polymorphism in the equine herpesvirus 1 (EHV-1) DNA polymerase gene (ORF30 A(2254) to G) has been associated with clinical signs of equine herpes myeloencephalopathy (EHM). The purpose of our study was to determine the odds ratio for this genetic marker and EHM using a panel of field isolates from North America collected over the past twenty-three years. EHV-1 isolates cultured at the Cornell University Animal Health Diagnostic Laboratory from 1984 to 2007 were retrieved along with their clinical histories. DNA was extracted from these EHV-1 cultures and allelic discrimina...
Cappelli K, Felicetti M, Capomaccio S, Pieramati C, Silvestrelli M, Verini-Supplizi A.The stress response is a critical factor in the training of equine athletes; it is important for performance and for protection of the animal against physio-pathological disorders.In this study, the molecular mechanisms involved in the response to acute and strenuous exercise were investigated using peripheral blood mononuclear cells (PBMCs). Results: Quantitative real-time PCR (qRT-PCR) was used to detect modifications in transcription levels of the genes for matrix metalloproteinase-1 (MMP-1) and interleukin 8 (IL-8), which were derived from previous genome-wide expression analysis. Signific...
Huang H, Lavoie-Lamoureux A, Lavoie JP.The endothelium plays a critical role in regulating leukocyte recruitment and migration during inflammation. Recent studies provide evidence that acetylcholine (ACh) and other cholinergic mediators block endothelial cells activation and leukocyte recruitment during inflammation. We thus postulated that the non-neuronal cholinergic system might modulate the recruitment of neutrophils during allergic pulmonary inflammation. In the present study, we examined the effects of cholinergic stimulation on the expression of neutrophil chemokines and adhesion molecules by endothelial cells stimulated by ...
Figueiredo MD, Salter CE, Andrietti AL, Vandenplas ML, Hurley DJ, Moore JN.Quantification of gene expression using real-time reverse transcription quantitative PCR (RT-qPCR) is a reliable method to monitor cellular responses to pro-inflammatory stimuli. The main objective of this study was to validate a set of equine primer pairs that can be routinely used to monitor expression of genes that are central to inflammatory and immune responses. This paper describes the steps used to optimize and validate 29 equine primer pairs for RT-qPCR assays using SYBR Green detection. To validate these assays, monocytes were isolated from three horses and stimulated with Escherichia...
Furet JP, Firmesse O, Gourmelon M, Bridonneau C, Tap J, Mondot S, Doré J, Corthier G.Pollution of the environment by human and animal faecal pollution affects the safety of shellfish, drinking water and recreational beaches. To pinpoint the origin of contaminations, it is essential to define the differences between human microbiota and that of farm animals. A strategy based on real-time quantitative PCR (qPCR) assays was therefore developed and applied to compare the composition of intestinal microbiota of these two groups. Primers were designed to quantify the 16S rRNA gene from dominant and subdominant bacterial groups. TaqMan probes were defined for the qPCR technique used ...
Wilson WC, Letchworth GJ, Jiménez C, Herrero MV, Navarro R, Paz P, Cornish TE, Smoliga G, Pauszek SJ, Dornak C, George M, Rodriguez LL.Sporadic outbreaks of vesicular stomatitis (VS) in the United States result in significant economic losses for the U.S. livestock industries because VS is a reportable disease that clinically mimics foot-and-mouth disease. Rapid and accurate differentiation of these 2 diseases is critical because their consequences and control strategies differ radically. The objective of the current study was to field validate a 1-tube multiplexed real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assay for the rapid detection of Vesicular stomatitis New Jersey virus and Vesicular st...
Nieto JE, Aleman M.Anesthetic-induced malignant hyperthermia has been documented in Quarter Horses and is caused by a single-point mutation in the ryanodine receptor 1 gene at nucleotide C7360G generating a R2454G amino acid substitution. An accurate, faster molecular test that is less prone to contamination would facilitate screening for the mutation in horses intended for breeding, in those undergoing surgical procedures, and in those with clinical signs compatible with malignant hyperthermia. Objective: To report a rapid and accurate method for the detection of the ryanodine receptor 1 C7360G mutation. Method...
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...
Miragliotta V, Lefebvre-Lavoie J, Lussier JG, Theoret CL.Horses suffer from a debilitating impediment in repairing wounds located on the lower limb that leads to the development of a fibroproliferative disorder (exuberant granulation tissue). This condition is a source of wastage since it often forces retirement from competition. Treatments that resolve or prevent this condition are still lacking, maybe due to deficient knowledge of the underlying molecular mechanisms. Fibroblast-to-myofibroblast conversion is an essential step allowing contraction during wound repair and is accompanied by an increase in OB-cadherin expression. Objective: To clone e...
Svansson V, Roelse M, Olafsdóttir G, Thorsteinsdóttir L, Torfason EG, Torsteinsdóttir S.Horses are hosts to two types of gammaherpesviruses, equine herpes virus (EHV) 2 and 5. While EHV-2 is ubiquitous in adult horses, EHV-5 has been less frequently described. Due to strong serological cross-reactivity, EHV-2 and -5 cannot be discriminated in broad spectrum antibody tests and are thus commonly referred to as gamma-EHV. Total IgG and IgG subclass response against gamma-EHV were determined in serum from 41 healthy Icelandic horses, thereof 20 adults, 10 foals aged 10 months, and 11 foals aged 1-4 months. Additionally, in 10 of the adult horses, interferon (IFN)-gamma and interleuki...
Zaayman D, Human S, Venter M.In recent years, West Nile virus has been responsible for outbreaks in regions where it has not previously been found. Five genetic lineages with specific geographic distributions exist. Recent outbreaks of WNV associated with the introduction of lineage 1 strains into the western hemisphere, together with the emergence of lineage 2 WNV in Central Europe, has highlighted the potential for spread of pathogenic WNV strains beyond their expected geographical boundaries. Therefore, genotyping of WNV strains may have important applications in surveillance and epidemiology. We report here the develo...
Riihimäki M, Raine A, Elfman L, Pringle J.Airborne factors in a conventionally managed racing stable and markers of pulmonary inflammation in the stabled horses were investigated on 3 occasions at 6-month intervals, including 2 winter periods and the intervening summer period. The stable measurements included inside and outside ambient temperature and relative humidity, levels of total and respirable dust, endotoxin, and 1,3-beta-glucan. Horses (n = 12) were examined in detail clinically as well as by endoscopy, bronchoalveolar lavage (BAL) cytology, and real-time polymerase chain reaction (RT-PCR) analysis of BAL-cells for IL-6 and I...
Miragliotta V, Lussier JG, Theoret CL.Healing of wounds located on the distal limbs of horses is often complicated by retarded epithelialization and the development of exuberant granulation tissue (proud flesh). Treatments that definitively resolve this pathological process are still unavailable. Molecular studies of the repair mechanism might contribute to the development of new therapeutic strategies. The study presented herein aimed to clone the full length cDNA and to study the spatio-temporal expression profile of mRNA and protein for LAMR1, previously attributed a role in wound epithelialization, during the repair of body an...
Foord AJ, Selleck P, Colling A, Klippel J, Middleton D, Heine HG.Equine influenza (EI) virus (H3N8) was identified in the Australian horse population for the first time in August 2007. The principal molecular diagnostic tool used for detection was a TaqMan real-time reverse transcription-polymerase chain reactions (RT-PCR) assay specific for the matrix (MA) gene of influenza virus type A (IVA). As this assay is not specific for EI, we developed a new EI H3-specific TaqMan assay targeting the haemagglutinin (HA) gene of all recent EI H3 strains. The IVA and the EI H3 TaqMan assays were assessed using in vitro transcribed RNA template, virus culture, diagnost...
Mori A, De Benedictis P, Marciano S, Zecchin B, Zuin A, Zecchin B, Capua I, Cattoli G.Equine rhinitis A and B viruses (ERAV and ERBV) are respiratory viruses of horses belonging to the family Picornaviridae. Although these viruses are considered to cause respiratory disease in horses and are potentially infectious for humans, little is known about their prevalence and pathogenesis. Virus isolation is often unsuccessful due to their inefficient growth and lack of cytopathic effect in cell cultures. Therefore, molecular assays should be considered as the method of choice to detect infection in symptomatic or apparently healthy horses. In the present study, a novel real-time duple...
Coyne MJ, Cousin H, Loftus JP, Johnson PJ, Belknap JK, Gradil CM, Black SJ, Alfandari D.Equine laminitis is a debilitating disease affecting the digital laminae that suspend the distal phalanx within the hoof. While the clinical progression of the disease has been well documented, the molecular events associated with its pathogenesis remain largely unknown. Using real time quantitative PCR (RT-qPCR), we have investigated the expression of genes coding for proteins containing a Disintegrin and Metalloprotease domain (ADAM), as well as genes encoding the natural inhibitors of these enzymes (tissue inhibitor of metalloprotease; TIMP) in horses with naturally-acquired (acute, chronic...
Stewart AJ, Pettigrew A, Cochran AM, Belknap JK.The liver and lung are not only described as "target organs" in sepsis in most species, but are purported to be sources of circulating inflammatory mediators central to the systemic inflammatory response syndrome (SIRS). As we have recently reported an inflammatory response in the laminar tissue in laminitis similar to that described in "target organs" in human sepsis, we investigated the inflammatory response of the lung and liver in the black walnut extract (BWE) model of equine laminitis to determine (1) if a similar systemic inflammatory response occurs in this laminitis model as described...
Mienaltowski MJ, Huang L, Stromberg AJ, MacLeod JN.Articular cartilage undergoes an important maturation process from neonate to adult that is reflected by alterations in matrix protein organization and increased heterogeneity of chondrocyte morphology. In the horse, these changes are influenced by exercise during the first five months of postnatal life. Transcriptional profiling was used to evaluate changes in articular chondrocyte gene expression during postnatal growth and development. Methods: Total RNA was isolated from the articular cartilage of neonatal (0-10 days) and adult (4-5 years) horses, subjected to one round of linear RNA ampli...
Jischa S, Walter I, Nowotny N, Palm F, Budik S, Kolodziejek J, Aurich C.To determine endometrial regeneration in postpartum mares by analysis of histologic features, apoptosis and cell proliferation markers, lectin binding, cytokines, and progesterone and estrogen receptors in endometrial biopsy specimens. Methods: 9 postpartum mares. Methods: Mares were examined on postpartum days 1, 9, and 16, and uterine biopsy specimens were obtained for histologic examination. Lectin binding was analyzed histochemically, and expressions of Ki-67 antigen (proliferation marker), lysozyme, and caspase 3 (apoptosis marker) were studied immunohistochemically. Gene expressions for ...
Pusterla N, Mapes S, Rejmanek D, Gebhart C.The objective of this study was to determine whether Lawsonia intracellularis was present in the feces of free-living animals collected on two equine premises with documented occurrence of equine proliferative enteropathy (EPE). Fresh feces from black-tailed jackrabbits (Lepus californicus, n=100), striped skunks (Mephitis mephitis, n=22), feral cats (Felis catus, n=14), Brewer's Blackbirds (Euphagus cyanocephalus, n=10), Virginian opossums (Didelphis virginiana, n=9), raccoons (Procyon lotor, n=4), California ground squirrels (Spermophilus beecheyi, n=3), and coyotes (Canis latrans, n=2) were...
Bae JH, Ahn K, Nam GH, Lee CE, Park KD, Lee HK, Cho BW, Kim HS.The horse BMAL1 gene encodes the brain and muscle Arnt-like protein 1, which is a key regulator of circadian rhythmic systems in most organs and cells. The first exon of the horse-specific BMAL1 gene is produced by an exonization event of LINE3 (CR1) and SINE (MIR) was detected by bioinformatic analysis. Alternative variants generated by cassette exon event in various horse tissues were also detected by RT-PCR amplification and sequencing. The cDNA sequences of the horse transcripts (BMAL1a, BMAL1b) contain additional 21 bp and 71 bp fragments relative to horse BMAL1. Quantitative real-time RT...
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...
Qi T, Hu Y, Hu Z, Zhao S, Cullinane A, Lyons P, Gildea S, Wang X.Quantitation of virions is one of the important indexes in virological studies. To establish a sensitive and rapid quantitative detection method for equine arteritis virus (EAV), an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed by using two EAV nucleoprotein monoclonal antibodies (mAbs), 2B9 and 2B3, prepared in this study. After condition optimization, mAb 2B9 was used as the capture antibody, and HRP-labeled 2B3 was chosen as the detecting antibody. The AC-ELISA had a good standard curve when viral particles of the Bucyrus EAV strain were used as a reference stan...
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...
Saini S, Singha H, Siwach P, Tripathi BN.Interleukin (IL)-4 and IL-10 activate plethora of immune cells and induce the humoral immune response. However, recombinant version of horse IL-4 and IL-10 has not been investigated to understand their immunomodulating activities. This study aimed to produce recombinant horse mature IL-4 and IL-10 in . Immune-modulating activities of recombinant horse IL-4 and IL-10 were investigated in peripheral blood mononuclear cells (PBMCs). Methods: Equine PBMCs were stimulated with recombinant IL-4 and IL-10. A proliferation of PBMCs was measured by XTT assay and cytokines induction was measured by enzy...
Peretich AL, Abbott LL, Andrews FM, Dhar MS.To determine whether expression of mRNA for sodium-potassium adenosine-triphosphatase (NAKA) and sodium-hydrogen exchanger (NHE) in samples of the nonglandular portion of the equine gastric mucosa was altered by exposure to volatile fatty acids (VFAs) in an acidic environment. Methods: 10 horses (5 or = 12 years old). Methods: Samples of the nonglandular portion of the gastric mucosa were collected and exposed in Ussing chambers to Ringer's solution (control samples), Ringer's solution containing a mixture of VFAs (pH, 1.5 or 4.0), or Ringer's solution containing acetic acid (pH, 1.5 or 4.0)....
Balasuriya UBR.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 econom...
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...
Herrera-Luna CV, Budik S, Helmreich M, Walter I, Aurich C.Glucocorticoids (GCs) as mediators of the stress response may affect Leydig cell function by inhibiting either luteinizing hormone receptor expression or testosterone biosynthesis. The isozymes 11β-hydroxysteroid dehydrogenase (11βHSD) 1 and 11βHSD2 control the intracellular cortisol levels. Little is known about the effects of stress on fertility in the equine. The objective of the present study was to determine the presence and cellular localization of glucocorticoid receptors (GCR) and glucocorticoid-metabolizing enzymes (11βHSD1 and 11βHSD2) in equine epididymal and testicular tissue ...
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...
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...
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...
Alnaeem AA, Hemida MG.Equine infectious anemia virus (EIAV) is one of the most important threats to the equine industry globally. This is due to the poor performance of the affected horses, which requires euthanization of the infected animals upon the infection confirmation. Infected animals remain carriers throughout their life. EIAV infection has been reported in many parts of the world, including North America, Europe, Asia, and Africa. However, the EIAV status is never assessed in horses in the Gulf area, especially in the Kingdom of Saudi Arabia (KSA). Objective: This study aimed to perform molecular and serol...
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...
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...
Nemoto M, Okita N, Kitahata M, Bannai H, Tsujimura K, Kinoshita Y, Kambayashi Y, Cullinane A, Yamanaka T, Ohta M.A rapid and sensitive diagnostic method is needed to help prevent the spread of equine influenza virus. The cobas Influenza A/B & RSV test for the cobas Liat system (Roche Diagnostics) is based on real-time reverse transcription polymerase chain reaction and is designed to broadly detect influenza A virus RNA within 20 minutes. It detected a broad range of equine influenza virus strains, and detected equine influenza virus RNA from nasal swabs of infected horses at the same level as real-time reverse transcription polymerase chain reaction, although it returned some invalid results (7.7%)...
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...
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...
Ellero N, Lanci A, Baldassarro VA, Alastra G, Mariella J, Cescatti M, Castagnetti C, Giardino L.Neonatal Encephalopathy (NE) may be caused by hypoxic ischemic insults or inflammatory insults and modified by innate protective or excitatory mechanisms. Understanding the underlying pathophysiology is important in formulating a rational approach to diagnosis. The preliminary aim was to clinically characterize a population of foals spontaneously affected by NE. The study aimed to: (i) evaluate nerve growth factor (NGF) and vascular endothelial growth factor (VEGF) levels in plasma samples obtained in the affected population at parturition from the mare’s jugular vein, umbilical cord vein an...
Craig AF, Packer GC, Guthrie AJ, Venter EH.A prospective study was undertaken during 2013 and 2014, to determine the prevalence of African horse sickness virus (AHSV) in Culicoides midges and the incidence of infection caused by the virus in 28 resident horses on two equine establishments on the East Rand, Gauteng Province, South Africa. Field caught Culicoides midges together with whole blood samples from participating horses were collected every two weeks at each establishment. Culicoides midges and blood samples were tested for the presence of AHSV RNA by real-time quantitative reverse transcription polymerase chain reaction. Nine i...
Ghaeli T, Karimi B, Kojouri GA, Dehkordi RR, Ahadi AM.One of the initial responses of the host's innate immunity of newborns against pathogens is the use of oxidative enzymes. This study aimed to evaluate changes in serum xanthine oxidoreductase (XOR) activity, the leukocyte myeloperoxidase (MPO) and XOR genes expression, and some biochemical parameters in healthy Darehshuri newborn foals up to 60 days of life. Blood samples were collected from 16 foals at 1, 7, 15, 30, and 60 days and used for detecting XOR activity, biochemical parameters, and also gene expression by real-time RT-PCR. High activity of XOR was observed at birth, explained by phy...
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...
Thirumalapura NR, Livengood J, Beeby J, Wang W, Goodrich EL, Goodman LB, Erol E, Tewari D.Neorickettsia risticii, an obligate intracellular bacterium, is the causative agent of Potomac horse fever (PHF). Diagnosis of PHF is based on demonstration of serum antibodies, isolation of N. risticii, and/or detection of nucleic acid by a PCR assay. An existing real-time PCR assay targeting the N. risticii 16S rRNA has been validated using blood samples from horses with colitis, and snails; to our knowledge, the performance of the assay for other sample types has not been reported. We describe here a modification of the 16S rRNA gene assay by the addition of a set of primers and probe targe...
Campbell MLH, Peachey L, Callan L, Wathes DC, de Mestre AM.This study was conducted to elucidate mare cervical dilation mechanisms by testing two hypotheses: (i) the proportion of collagen staining in histological samples of mare cervices and (ii) the abundance of hormone receptors in the equine cervix differ with stage of the oestrous cycle and site within the cervix. Tissues and jugular vein blood samples were collected from 15 mares. Collagen content was assessed using Masson's Trichome staining. Receptor abundance was assessed using RT-PCR, qRT-PCR and immunohistochemistry. In sub-epithelial stroma, there was less collagen during the follicular th...
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...
