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.
De Coster T, Van Poucke M, Bogado Pascottini O, Angel-Velez D, Van den Branden E, Peere S, Papas M, Gerits I, Govaere J, Peelman L, Vermeesch JR....In addition to fulfilling many breeders' curiosity, equine embryonic sex determination can have a profound commercial impact. However, the application of currently described assays for equine embryonic sexing has rendered variable diagnosis and validation rates, with sensitivity being the main problem. In addition, while pregnancy results of in vivo-flushed equine embryos following a needle aspiration biopsy equal those of non-biopsied embryos, the effect on in vitro-produced embryos is unknown. Here, we aimed to develop a highly sensitive and specific assay for equine sex determination that c...
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...
Carossino M, Balasuriya UBR, Thieulent CJ, Barrandeguy ME, Vissani MA, Parreño V.Equine rotavirus A (ERVA) is the leading cause of diarrhea in foals, with G3P[12] and G14P[12] genotypes being the most prevalent. Recently, equine G3-like RVA was recognized as an emerging infection in children, and a group B equine rotavirus (ERVB) was identified as an emergent cause of foal diarrhea in the US. Thus, there is a need to adapt molecular diagnostic tools for improved detection and surveillance to identify emerging strains, understand their molecular epidemiology, and inform future vaccine development. We developed a quadruplex TaqMan RT-qPCR assay for differentiation of ERVA an...
De Paolis L, Armando F, Montemurro V, Petrizzi L, Straticò P, Mecocci S, Guarnieri C, Pezzolato M, Fruscione F, Passeri B, Marruchella G, Razzuoli E.Vulvar squamous cell carcinoma (VSCC) has been recently associated with Equus caballus papillomavirus type 2 (EcPV2) infection. Still, few reports concerning this disease are present in the literature. Objective: To describe a case of naturally occurring EcPV2-induced VSCC, by investigating tumour ability in undergoing the epithelial-to-mesenchymal transition (EMT). Methods: Case report. Methods: A 13-year-old Haflinger mare was referred for a rapidly growing vulvar mass. After surgical excision, the mass was submitted to histopathology and molecular analysis. Histopathological diagnosis was c...
Morris ERA, Schroeder ME, Ferro PJ, Waller AS, McGlennon AA, Bustos CP, Gressler LT, Wu J, Lawhon SD, Boyle AG, Lingsweiler S, Paul N, Dimitrov K....Strangles is a contagious bacterial disease of horses caused by Streptococcus equi subspecies equi (SEE) that occurs globally. Rapid and accurate identification of infected horses is essential for controlling strangles. Because of limitations of existing PCR assays for SEE, we sought to identify novel primers and probes that enable simultaneous detection and differentiation of infection with SEE and S. equi subsp. zooepidemicus (SEZ). Comparative genomics of U.S. strains of SEE and SEZ (n = 50 each) identified SE00768 from SEE and comB from SEZ as target genes. Primers and probes for real-ti...
Weese JS, Saab M, Moore A, Cai H, McClure JT.To compare PCR and culture results for the detection of subspecies . Respiratory tract samples (N = 158) from horses being tested for Bacterial culture was carried out on samples from which was detected by quantitative real-time PCR. was isolated from 12 (7.6%) samples: 4/9 (44%) samples when the PCR cycle threshold (C) was ≤ 30, 7/30 (23%) when the C was 30.1 to 35, and 1/119 (0.8%) when the C was 35.1 to 40. The highest C sample from a sample that yielded a positive culture was 36.9. The optimal Youden's J value was at a C of 34.2, the same value as determined by number needed to misdi...
Fukuda K, Mita H, Tamura N, Kuroda T, Kuwano A, Takahashi T, Sato F.Persistent synovitis damages the articular cartilage in horses. To evaluate the effectiveness of treatment for synovitis using a model induced by intra-articular administration of monoiodoacetic acid (MIA), it is necessary to identify inflammatory biomarkers characteristic of the MIA model. Synovitis was induced by administering MIA into the unilateral antebrachiocarpal joints of five horses, and saline was injected into the contralateral joints as a control on day 0. Clinical and ultrasonographic examinations and synovial fluid collection were performed on days 0, 1, 2, 7, 14, 21, 28, and 35....
Craig NM, Munguia NS, Trujillo AD, Wilkes R, Dorr M, Marsella R.Preliminary evidence supports a role for IL-31 in equine insect bite hypersensitivity (IBH) and pruritus. Our studies investigated IL-31 and IL-31 receptor-α (IL-31RA) transcription in leukocytes from normal and IBH horses in response to Culicoides nubeculosus. 19 normal and 15 IBH horses were recruited in the summer of 2019 (low-dose study) and 8 normal and 10 IBH horses in the winter of 2022 to 2023 (high-dose study). Normal horses had no history or signs of allergic skin disease, while IBH horses had a history and clinical signs compatible with IBH. Pruritus was scored using a visual analo...
Gysens L, Martens A, Haspeslagh M.Bovine papillomavirus (BPV) types 1 and 2 are causally associated with equine sarcoid, the most common mesenchymal neoplasm of horses, but the viral load (VL) differs between lesions. Sensitive and accurate BPV detection and quantification is essential for clinicians to confirm clinical suspicion, as well as in research settings for stratifying these skin lesions. Due to the limitations of histopathology in sarcoid diagnosis, PCR screening of superficial swabs constitutes the principal sampling method for BPV detection. This study aimed to investigate the ability of superficial swabs and fine-...
Wong KS, Cheung HW, Szeto CWL, Tsang CYN, Wan TSM, Ho ENM.Illicit administration of transgene into horses is a form of gene doping that has been a key concern in equine sports. The large number of potential performance-enhancing transgenes has demanded a cost-effective and reliable detection method. Multiplex qPCR is a relevant technique, but the cross-talking between fluorophores and high background noise limits the method sensitivity and specificity. This study reports a simpler multiplexing approach by using the same fluorophore for four hydrolysis probes each targeting one of the four transgenes: human growth hormone, insulin-like growth factor 1...
Belacic ZA, Sullivan SN, Rice HC, Durgam SS.To investigate the effects of interleukin-1β (IL-1β) and methylprednisolone acetate (MPA) on equine intrabursal deep digital flexor tendon (DDFT) and navicular bone fibrocartilage (NBF) cells in vitro. Methods: Third passage DDFT and NBF cells from 5 healthy donor horses ages 11-17 years euthanized for reasons unrelated to musculoskeletal conditions. Methods: Aggregate cultures were incubated with culture medium alone (control), 10 ng/mL IL-1β, 10 ng/mL IL-1β + 0.05 mg/mL MPA, or 10 ng/mL IL-1β + 0.5 mg/mL MPA for 24 hours. Extracellular matrix (ECM) gene expressions were assessed via rea...
Beghelli D, Zallocco L, Angeloni C, Bistoni O, Ronci M, Cavallucci C, Mazzoni MR, Nuccitelli A, Catalano C, Hrelia S, L쫌hini A, Giusti L.Intense exercise can cause inflammation and oxidative stress due to the production of reactive oxygen species. These pathophysiological processes are interdependent, and each one can induce the other, creating a vicious circle. A placebo-controlled blind study was carried out in show jumping horses (n. 16) to evaluate the effects of a commercial dietary supplement (Dolhorse® N.B.F. Lanes srl, Milan, Italy) containing Verbascum thapsus leaf powder (1.42%), Curcuma longa (14.280 mg/kg), and Boswellia serrata (Roxb ex Colebr) (14.280 mg/kg) extracts. Before and after 10 days of dietary supplemen...
Riveroll AL, Skyba-Lewin S, Lynn KD, Mubyeyi G, Abd-El-Aziz A, Kibenge FST, Kibenge MJT, Cohen AM, Esparza-Gonsalez B, McD￮ L, Montelpare WJ.Adipose-derived stem cells (ADSCs) are used in tissue regeneration therapies. The objective of this study is to identify stable reference genes (RGs) for use in gene expression studies in a characterized equine adipose-derived mesenchymal stem cell (EADMSC) differentiation model. ADSCs were differentiated into adipocytes (ADs) or osteoblasts (OBs), and the proteomes from these cells were analyzed by liquid chromatography tandem mass spectrometry. Proteins that were stably expressed in all three cells types were identified, and the mRNA expression stabilities for their corresponding genes were ...
Zamith Cunha R, Zannoni A, Salamanca G, De Silva M, Rinnovati R, Gramenzi A, Forni M, Chiocchetti R.The metacarpophalangeal joint undergoes enormous loading during locomotion and can therefore often become inflamed, potentially resulting in osteoarthritis (OA). There are studies indicating that the endocannabinoid system (ECS) modulates synovium homeostasis, and could be a promising target for OA therapy. Some cannabinoid receptors, which modulate proliferative and secretory responses in joint inflammation, have been functionally identified in human and animal synovial cells. Unassigned: To characterize the cellular distribution of the cannabinoid receptors 1 (CB1R) and 2 (CB2R), and the can...
Wang J, Guo K, Li S, Liu D, Chu X, Wang Y, Guo W, Du C, Wang X, Hu Z.Salmonella enterica subsp. serovar Abortusequi is a major pathogen in horse and donkey herds, causing abortion in pregnant equids and resulting in enormous economic losses. A rapid and reliable method is urgently needed to detect . Abortusequi in herds where the disease is suspected. To achieve this goal, a TaqMan-based real-time PCR assay targeting the gene for the flagellin protein phase 2 antigen FljB was developed. This real-time PCR assay had high specificity, sensitivity, and reproducibility. The detection limit of the assay was 30 copies/μL of standard plasmid and 10 CFU/μL of bacter...
Petry S, Breuil MF, Duquesne F.Contagious equine metritis (CEM) detection by PCR is recognized by the European Union according to Commission Implementing Regulation (EU) No 846/2014, and real-time PCR is now recommended by the World Organisation for Animal Health Terrestrial Manual at the same level as the culture method. The present study highlights the creation of an efficient network of approved laboratories in France in 2017 for CEM detection by real-time PCR. The network currently consists of 20 laboratories. A first proficiency test (PT) was organized by the national reference laboratory for CEM in 2017 to evaluate th...
Turner S, Knych HK, Adams AA.Cannabidiol (CBD) has potential to reduce pain and inflammation in humans leading to the interest of use in equine. The purpose of this study was to determine the effects of CBD on immune function by measuring inflammatory cytokines and antibody responses to vaccination, as well as other health parameters in senior horses. Horses were orally-dosed with CBD (2 mg/kg: 13 horses) or control (soy oil: 14 horses) daily for 90 days, from July 2021 to November 2021. Peripheral blood samples were collected on days 0, 30, 60, and 90 before administering treatments. On day 90 all horses were kept on tr...
Patroca da Silva S, Barbosa de Almeida Medeiros D, Ribeiro Cruz AC, Marques França AF, Diniz Nunes BT, Guerreiro Rodrigues DS, Pinto da Silva EV....In 2018, during the surveillance for West Nile virus (WNV) in horses with neurological clinical signs in the state of Espírito Santo (Brazil), 19 animals were investigated, and 52 biological samples were collected for WNV diagnostic. One brain sample was positive for WNV by RT-qPCR and the virus was isolated in C6/36 cell culture and sequenced. We obtained a nearly complete genome of WNV co-infected with Peruvian horse sickness virus (PHSV) in the cell culture. After confirmation of PHSV by next-generation sequencing, a new PHSV RT-qPCR protocol was developed, which was used to detect another...
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...
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...
Tiwari M, Sodhi M, Verma P, Vivek P, Kataria RS, Niranjan SK, Bharti VK, Masharing N, Gujar G, Chanda D, Mukesh M.The identification of appropriate references genes is an integral component of any gene expression-based study for getting accuracy and reliability in data interpretation. In this study, we evaluated the expression stability of 10 candidate reference genes (GAPDH, RPL4, EEF1A1, RPS9, HPRT1, UXT, RPS23, B2M, RPS15, ACTB) in peripheral blood mononuclear cells of livestock species that are adapted to high altitude hypoxia conditions of Leh-Ladakh. A total of 37 PBMCs samples from six native livestock species of Leh-Ladakh region such as Ladakhi cattle, Ladakhi yak, Ladakhi donkey, Chanthangi goat...
Wang X, Asgenbaatar N, Shen Y, Yi M, Zhao B, Ren H, Davshilt T, Ulaangerel T, Wang M, Burenbaatar A, Tian S, Li B, Dugarjav M, Bou G.Proliferation of embryonic fibroblasts under the same cell culture conditions, hinny embryonic fibroblasts (HiEFs) was slower than horse embryonic fibroblast (HEFs), donkey embryonic fibroblasts (DEFs) and mule embryonic fibroblasts (MuEFs). The imprinted genes IGF2 and IGF2R are important for cell proliferation. Therefore, we investigated whether the slower proliferation of HiEFs is related to an aberrant gene expression of IGF2 or its receptors or genes influencing the expression of the IGF2 system. Results: Real-time polymerase chain reaction, immunofluorescence and cell starving experiment...
Diel de Amorim M, Bramer SA, Rajamanickam GD, Klein C, Card C.Our objectives were to examine changes in endometrial and luteal gene expression during estrus, diestrus, pregnancy and treatments to induce luteolysis and putatively induce luteostasis. Groups were: Diestrus (DIEST), Estrus (ESTR), Pregnant (PREG), Oxytocin (OXY), Carbetocin (CARB), and Meclofenamic acid (MFA). Blood was obtained from day (D)12 to D15 for measurement of oxytocinase, also referred to as leucyl-cysteinyl aminopeptidase (LNPEP) and progesterone. Luteal biopsies were obtained on D12 and D15 and an endometrial biopsy on D15. Real-time RT-PCR was performed for the following genes: ...
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...
Cao X, Qiu X, Shi N, Ha Z, Zhang H, Xie Y, Wang P, Zhu X, Zhao W, Zhao G, Jin N, Lu H.Getah virus (GETV) is a mosquito-borne, single-stranded, positive-sense RNA virus belonging to the genus of the family . Natural infections of GETV have been identified in a variety of vertebrate species, with pathogenicity mainly in swine, horses, bovines, and foxes. The increasing spectrum of infection and the characteristic causing abortions in pregnant animals pose a serious threat to public health and the livestock economy. Therefore, there is an urgent need to establish a method that can be used for epidemiological investigation in multiple animals. In this study, a real-time reverse tr...
De Paolis L, De Ciucis CG, Peletto S, Cappelli K, Mecocci S, Nervo T, Guardone L, Crescio MI, Pietrucci D, Fruscione F, Gabbianelli F, Turco S....Papillomavirus (PV) infections may be related to anogenital lesions and cancer development in humans and several other animal species. To date, 11 different PVs have been reported in horses. Among them, a newly described PV named Papillomavirus Type9 (EcPV9) was thus far only reported in the semen of a stallion with penile lesions in Australia. This study reports for the first time the presence of EcPV9 in asymptomatic Italian horses. From July 2020 to January 2022, genital brush samples were collected from 209 horses with no apparent signs of neoplastic disease and no PV-associated lesions, ...
Fürer F, Fraefel C, Lechmann J.Equid gammaherpesvirus 2 (EHV-2) and 5 (EHV-5) are widely distributed in the equines. Although their pathogenic potential is not yet fully understood, they appear to play a role in disease patterns like equine multinodular pulmonary fibrosis. In this study, a multiplex real-time PCR (rtPCR) was designed to detect DNA of the glycoprotein H (EHV-2) and E11 gene (EHV-5). Analytical specificity was determined by testing DNA of other herpesviruses by SYBR Green rtPCR and melting curve analysis, as well as Sanger sequencing of positive field samples. Analytical sensitivity was assessed by stand...
Kornicka-Garbowska K, Groborz S, Lynda B, Galuppo L, Marycz K.Synovitis (SI) is one of the most common and serious orthopedic diseases in horses of different age, breed and sex, which contributes to the development of osteoarthritis. The burden of SI includes economic loss and represents a real challenge for current veterinary health care. At the molecular level, fibroblasts-like synoviocytes (FLS) are recognized as major cell populations involved in SI pathogenesis. In the course of SI, FLSs are losing their protective and pro-regenerative cytological features, become highly proliferative and initiate various stress signaling pathways. Fibroblast-like s...
Diel de Amorim M, Bramer SA, Rajamanickam GD, Klein C, Card C.Our understanding of the temporal changes in endometrial and luteal gene transcripts related to the actions of oxytocin and prostaglandin during early equine pregnancy is incomplete. Additionally, the role of oxytocinase, also known as Leucyl-cystinyl aminopeptidase (LNPEP), during early pregnancy in mares has not been previously investigated. Luteal and endometrial biopsies were obtained on Day (D)8, D10, D12 and D15 post-ovulation in pregnant (PREG) and diestrus (DIEST) mares for real-time qPCR. Differences in endometrial gene expression occurred over time in: SLC2A4, SLC2A1, PTGES, OXTR and...
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...
Davis MS, Malayer JR, Vandeventer L, Royer CM, McKenzie EC, Williamson KK.Athletes who perform repeated exercise while breathing cold air have a high prevalence of asthmalike chronic airway disease, but the mechanism linking such activity to airway inflammation is unknown. We used a novel animal model (exercising horses) to test the hypothesis that exercise-induced chronic airway disease is caused by exposure of intrapulmonary airways to unconditioned air, resulting in the upregulation of cytokine expression. Bronchoalveolar lavage fluid (BALF) was obtained from eight horses 5 h after submaximal exercise while they breathed room temperature or subfreezing air in a r...
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...
Donnelly KS, Giuliano EA, Sharm A, Mohan RR.To explore the effect of suberoylanilide hydroxamic acid (SAHA) (i) on corneal fibroblast differentiation, morphology, and viability; and (ii) on the expression levels of matrix metalloproteinases (MMPs) 2 and 9 using an in vitro model of equine corneal fibrosis. Methods: Healthy donor corneas were used to generate primary cultures of equine corneal fibroblasts. The fibroblasts were exposed to 5 ng/mL TGFβ1 to induce myofibroblast formation. The cultures were treated with either 5 μm or 10 μm SAHA for 72 h in the presence of TGFβ1. Real-time PCR and immunocytochemistry were used to determi...
Padoan E, Ferraresso S, Pegolo S, Castagnaro M, Barnini C, Bargelloni L.The goal of the present study was to investigate mRNA expression levels of several cytokines and inflammatory mediators in broncho-alveolar lavage (BAL) fluid and respiratory epithelium in recurrent airway obstruction (RAO)-affected horses. RAO, also called heaves, is a common, performance-limiting, equine respiratory disease with clinical signs and pathophysiological similarities to human asthma, and characterized by bronchospasm, neutrophilic infiltration and increased mucus in the airways. Six RAO-affected horses were examined twice within 15 days and seven clinically healthy horses were ex...
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...
Fumuso E, Giguère S, Wade J, Rogan D, Videla-Dorna I, Bowden RA.Endometrial mRNA expression of the pro-inflammatory cytokines interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) was assessed in mares resistant (RM) or susceptible (SM) to persistent post-breeding endometritis (PPBE). Eight RM and eight SM, were selected based on reproductive records and functional tests out of a herd of 2,000 light cross-type mares. Three experiments were done to study transcription patterns in (i) basal conditions; (ii) after artificial insemination (AI); and (iii) after administration of an immunomodulator at time of artificial ...
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...
Nieto JE, MacDonald MH, Braim AE, Aleman M.Horses are exquisitely sensitive to bacterial endotoxin and endotoxaemia is common in colic cases. In this study, gene expression of inflammatory cytokines was characterised in the blood of healthy horses following i.v. administration of lipopolysaccharide (LPS). Six horses received an LPS infusion and 6 controls received an equivalent volume of saline. Gene expression of genes encoding interleukin (IL)-1alpha, IL-1beta, IL-6, IL-8, and tumour necrosis factor-alpha (TNF-alpha) was quantified by real-time PCR. Gene expression of all inflammatory cytokines was upregulated following administratio...
Knight CG, Dunowska M, Munday JS, Peters-Kennedy J, Rosa BV.Equus caballus papillomavirus type 2 (EcPV-2) infection has been associated with equine genital squamous cell carcinomas (SCCs). However, quantitative PCR (qPCR) has not been performed to determine viral copy numbers within these lesions. Additionally, the frequency with which EcPV-2 can be detected in other common sites of equine SCC development remains uncertain. The aim of this study was to develop a qPCR assay to estimate the viral load in a variety of equine tissue samples. These included 40 SCC lesions, 19 penile non-SCC or precursor disease lesions, and 222 tissues without observable le...
Cappelli K, Felicetti M, Capomaccio S, Nocelli C, Silvestrelli M, Verini-Supplizi A.Athletic performance is both a stress factor and an adaptive response to exercise that may be modulated by training, reduce inflammation and help prevent disease. Studies on the endocrinology of exercise and training have demonstrated the existence of an integrated metabolic network of hormone and cytokine regulation. Subsequent molecular studies have shown that repeated bouts of exercise may establish new basal levels of gene expression at rest. The Thoroughbred horse may be a useful 'exercise model' for inter-individual comparisons between subjects with homogeneous genetic and environmental ...
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...
Bogaert L, Van Poucke M, De Baere C, Dewulf J, Peelman L, Ducatelle R, Gasthuys F, Martens A.Equine sarcoids, the most common skin tumours in horses, are induced by bovine papillomavirus (BPV). Their clinical appearance varies from small stable patches to aggressively growing masses. Differences in BPV load and mRNA expression and Ki67 and p53 immunostaining among four clinical types (fibroblastic, occult, nodular and verrucous sarcoids) were evaluated to test the hypothesis that the clinical behaviour of equine sarcoids correlates with BPV activity. Viral load and expression of the BPV E2, E5, E6 and E7 genes were determined using quantitative real-time PCR. The proliferative fractio...
Agüero M, Gómez-Tejedor C, Angeles Cubillo M, Rubio C, Romero E, Jiménez-Clavero A.African horse sickness is an arthropod-borne disease of the equine included in the World Organization for Animal Health (OIE) list with important economic consequences for horse trade. The disease is caused by African horse sickness virus (AHSV; family Reoviridae, genus Orbivirus), which is transmitted by Culicoides midges. It is endemic in sub-Saharan Africa, spreading occasionally outside this area where the occurrence of Culicoides vectors allows virus transmission. Currently, only conventional (gel-based) reverse transcription polymerase chain reaction (RT-PCR) protocols are available for ...
Lowe W, March JK, Bunnell AJ, O'Neill KL, Robison RA.Methods for the rapid detection and differentiation of the Burkholderia pseudomallei complex comprising B. pseudomallei, B. mallei, and B. thailandensis, have been the topic of recent research due to the high degree of phenotypic and genotypic similarities of these species. B. pseudomallei and B. mallei are recognized by the CDC as tier 1 select agents. The high mortality rates of glanders and melioidosis, their potential use as bioweapons, and their low infectious dose, necessitate the need for rapid and accurate detection methods. Although B. thailandensis is generally avirulent in mammals, ...
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...
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...
Perglione CO, Gildea S, Rimondi A, Miño S, Vissani A, Carossino M, Cullinane A, Barrandeguy M.In 2012, equine influenza (EI) virus was confirmed as the cause of outbreaks of respiratory disease in horses throughout South America. In Uruguay and Argentina, hundreds of vaccinated thoroughbred horses in training and racing facilities were clinically affected. Objective: To characterise the EI viruses detected during the outbreak in Uruguay and Argentina. Methods: Virus was detected in nasopharyngeal swabs by a pan-reactive influenza type A real-time RT-PCR. The nucleotide sequence of the HA1 gene was determined and analysed phylogenetically using mega 5 software. Amino acid sequences alig...
Gather T, Walter S, Todt D, Pfaender S, Brown RJP, Postel A, Becher P, Moritz A, Hansmann F, Baumgaertner W, Feige K, Steinmann E, Cavalleri JV.Non-primate hepacivirus (NPHV), a recently discovered hepatotropic virus infecting horses, is phylogenetically the closest known homologue of hepatitis C virus (HCV). The main route for acquiring HCV infection in childhood is vertical transmission. However, nothing is known about the natural mode of transmission for NPHV. To investigate the possibility of vertically transmitted NPHV infection in horses, 20 Thoroughbred broodmares and their foals were monitored during foaling season 2015 until 6 months post-partum. Prepartal serum was taken from the mares, and during foaling umbilical cord bloo...
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...
Guthrie AJ, Maclachlan NJ, Joone C, Lourens CW, Weyer CT, Quan M, Monyai MS, Gardner IA.Blood samples collected from 503 suspect cases of African horse sickness (AHS) and another 503 from uninfected, unvaccinated South African horses, as well as 98 samples from horses from an AHS free country, were tested with an AHS virus (AHSV) specific duplex real-time reverse transcription quantitative PCR (RT-qPCR) assay and virus isolation (VI). The diagnostic sensitivity and specificity of this AHSV RT-qPCR assay and VI were estimated using a 2-test 2-population Bayesian latent class model which made no assumptions about the true infection status of the tested animals and allowed for the p...
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 ...
Peckle M, Pires MS, Dos Santos TM, Roier EC, da Silva CB, Vilela JA, Santos HA, Massard CL.The aim of this study was to detect Theileria equi (Laveran 1901) DNA in horses and ticks using real-time PCR and to list the factors associated with infection in animals located in the Seropedica and Petropolis municipalities of the state of Rio de Janeiro. We tested blood samples from 314 horses and samples from 300 ticks, including 191 Amblyomma cajennense, 104 Dermacentor nitens, and 5 Ixodida larvae. Factors inherent to the horse, the ownership, and animal management were obtained from an epidemiological questionnaire and were evaluated in association with the presence of T. equi DNA in t...
Kurowski PB, Traub-Dargatz JL, Morley PS, Gentry-Weeks CR.To use real-time polymerase chain reaction (PCR) technology to develop a highly sensitive and specific diagnostic assay for the detection of Salmonella spp in fecal specimens. Methods: 299 fecal specimens from cattle, horses, and dogs. Methods: Enrichment of fecal specimens was followed by genomic DNA extraction by use of commercially available isolation kits. Real-time PCR assay was performed to target a Salmonella spp-specific DNA segment. Results of real-time PCR assay were compared with bacterial culture results to determine relative sensitivity and specificity. Results: Use of the spaQ pr...
Peffers MJ, Milner PI, Tew SR, Clegg PD.SOX9 is a transcription factor that is essential for cartilage extracellular matrix (ECM) formation. Osteoarthritis (OA) is characterised by a loss of cartilage ECM. In chondrocytes SOX9 gene expression is regulated by osmotic loading. Here we characterise SOX9 mRNA regulation through static and cyclical application of hyperosmotic conditions in normal and OA monolayer equine chondrocytes. Furthermore, we investigate whether extracellular signal-regulated protein kinase (ERK)1/2 mitogen-activated protein kinases (MAPK) pathways have a role in this regulation of SOX9. Methods: Equine chondrocyt...
Vieira RF, Vidotto O, Vieira TS, Guimaraes AM, Santos AP, Nascimento NC, Santos NJ, Martins TF, Labruna MB, Marcondes M, Biondo AW, Messick JB.The aims of this study were to determine the prevalence of hemoplasmas in a rural Brazilian settlement's population of human beings, their dogs and horses, highly exposed to tick bites; to identify the tick species parasitizing dogs and horses, and analyze factors associated with their infection. Blood samples from 132 dogs, 16 horses and 100 humans were screened using a pan-hemoplasma SYBR green real-time PCR assay followed by a species-specific TaqMan real-time PCR. A total of 59/132 (44.7%) dog samples were positive for hemoplasmas (21 Mycoplasma haemocanis alone, 12 ' Candidatus Mycoplasma...
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...
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...
Davies RS, Madigan JE, Hodzic E, Borjesson DL, Dumler JS.Anaplasma phagocytophilum is the zoonotic cause of granulocytic anaplasmosis. We hypothesized that immune response, specifically gamma interferon (IFN-γ), plays a role in disease severity. To test this, horses were infected and IFNG expression was pharmacologically downregulated using corticosteroids. Eight horses were infected with A. phagocytophilum; 4 received dexamethasone on days 4 to 8 of infection. Clinical signs, hematologic parameters, and transcription of cytokine/chemokine genes were compared among treated and untreated horses. Infection was quantitated by msp2 real-time PCR and mi...
Leon A, Richard E, Fortier C, Laugier C, Fortier G, Pronost S.Abortion, stillbirth and neonatal death are major causes of equine mortality and cause severe economic loss to the equine industry. The present study was based on a complete necropsy protocol associated with classical microbiological examinations and molecular biology on 407 cases of abortion, stillbirths and neonate death. Based on this retrospective survey, "less common" abortive infectious agents were characterised by molecular tools in nine independent cases of abortion or neonate mortality. Among others, Chlamydophila abortus (1 case), Coxiella burnetii (6 cases) and Neospora caninum (3 c...
Klein C, Troedsson MH, Rutllant J.The process of water movement in the excurrent duct system of the male reproductive tract is pivotal for establishment of male fertility. The objective was to elucidate expression of aquaporin (AQP) water channels in the stallion reproductive tract. Real-time RT-PCR detected expression of AQP0-5 and AQP7-11 in testis, epididymis, and ductus deferens of mature stallions. There were two main expression patterns: (1) higher expression in testis than in epididymis and ductus deferens (AQP0, -4, -5, -8, -10, and -11); and (2) lower expression in testis than in epididymis and ductus deferens (AQP1, ...
