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.
Myćka G, Ropka-Molik K, Cywińska A, Stefaniuk-Szmukier M.Among numerous genes that have been a focus of equine genetic research, the (Klotho) and (Alpha-actinin-3) genes stand out due to their significant roles in muscle function and overall health, as well as performance ability. Previous studies on Arabian horses and other mammalians have shown that both and occur in different isoforms that seem to have different roles in metabolism. The main purpose of this present study was to describe different isoforms (, , , , , ) expression levels affected by the endurance effort in Arabian horses. Methods: Blood samples were taken from a group of = 10 ...
McCreight KA, Barbosa LN, Odoi A, Reed P, Rajeev S.We estimated the seroprevalence in dogs, cats, and horses from Tennessee, USA, using the microscopic agglutination test (MAT) against 12 serovars. We observed seropositivity in 110 of 374 (29.4%) dogs, 21 of 170 (12.4%) cats, and 42 of 88 (47.7%) horses. The highest seroprevalence was observed for serovars Autumnalis (74.6%) in dogs, and Bratislava in cats (42.9%) and horses (95.2%). We found a significant level of potential cross-reactivity between multiple serovars tested, with highest cross-reactivity to serovar Autumnalis in dogs. seroprevalence was significantly higher in vaccinated ...
is a tick-borne hemoparasite that causes equine piroplasmosis. It has a significant economic impact, decreasing performance and affecting animal welfare. This study aimed to identify DNA in the blood of horses from households in the southwestern and western regions of Romania. We included 310 animals, from which blood was collected via EDTA. To test the samples for the parasite genome, we used real-time PCR and conventional PCR. The prevalence of was 5.81% (18/310) in apparently healthy horses, suggesting that this parasite is enzootic in the regions studied, although veterinarians did not...
Cardeti G, Manna G, Cersini A, Nardini R, Rosati S, Reina R, Cittadini M, Sittinieri S, Altigeri A, Marcario GA, Scicluna MT.The mechanisms of the innate immunity control of equine infectious anemia virus in horses are not yet widely described. Equine monocytes isolated from the peripheral blood of three Equine infectious anemia (EIA) seronegative horses were differentiated in vitro into macrophages that gave rise to mixed cell populations morphologically referable to M1 and M2 phenotypes. The addition of two equine recombinant cytokines and two EIA virus reference strains, Miami and Wyoming, induced a more specific cell differentiation, and as for other species, IFNγ and IL4 stimulation polarized horse macrophages...
Migdał A, Migdał Ł, Oczkowicz M, Tombarkiewicz B, Okólski A.Colostrum contains substances such as hormones, enzymes, polyamides, nucleic acid derivatives, and amino acid derivatives and also includes immunomodulating substances, proline-rich polypeptides, immunoglobulins (Ig) as well as bacteriostatic compounds. The aim of the study was to evaluate the impact of colostral Ig on the level of expression of selected Toll-like receptor genes (TLR3, TLR4, and TLR7). The experiments were conducted on 25 Polish Pony foals. Blood samples were collected according to the following scheme: before first suckling, at the 1st, 3rd, 5th, 10th, 20th, and 30th, day of ...
Toft K, Honoré ML, Ripley NE, Nielsen MK, Fromm B, Mardahl M, Nielsen LN, Nejsum P, Thamsborg SM, Cirera S, Pihl TH.The equine bloodworm, Strongylus vulgaris, is a highly pathogenic parasite causing potentially fatal vascular and intestinal damage. Parasites express and release microRNAs (miRNAs) for internal regulation and to modulate host immunity. The complete set of miRNAs expressed by S. vulgaris (the S. vulgaris miRNAome) remains unannotated and the aim of this study was to annotate the miRNAome of L4 and L5 stages of S. vulgaris, and to examine differences in miRNA abundance between larval stages and sexes. Furthermore, we aimed to determine if miRNAs were detectable in excretory/secretory products (...
Sreenivasan CC, Naveed A, Uprety T, Soni S, Jacob O, Adam E, Wang D, Li F.Using metagenomic sequencing we identified equine rotavirus group B (ERVB) of ruminant origin in foal diarrhea outbreaks in the 2021 foaling season. To further investigate ERVB occurrence and determine its environmental stability, we collected mare and foal fecal samples from different farms in Central Kentucky during the 2022 foaling season. The RT-qPCR-based analyses showed that ERVB genome was detected in 16.67 % (42/252) of surveyed mare samples and 26.56 % (34/128) of foal samples. Furthermore, 94.12 % (16/17) of collected soil samples and 100 % (13/13) of water samples obtained f...
Cerveira-Pinto M, Wójtowicz A, Pires MA, Kordowitzki P, Skarzynski D, Ferreira-Dias G, Szóstek-Mioduchowska A, Amaral A.Increased synthesis and deposition of collagen (COL) in the extracellular matrix (ECM) of equine endometrium contributes to endometrosis. Toll-like receptors (TLRs) are transmembrane receptors involved in the innate immune response, recognized for their role in antigen recognition and previously associated with equine endometritis. The TLRs not only recognize pathogen-associated molecular patterns but also regulate inflammations, fibrosis and cancer. The aim of this study was to explore the relationship between TLR expression at different stages of Kenney and Doig's (K-D) grading and COL1 exp...
de Oliveira UV, Varjão JL, de Jesus Deiró AG, Maciel BM, Silva FL, Pinheiro AM, Gondim LFP, Munhoz AD.The aim of this study was to isolate from equids destined for slaughter in a Brazilian slaughterhouse. A total of 354 equids were analyzed, with blood samples collected from all the animals and samples of masseter muscle and brain tissue collected from 319 animals. A serological test was conducted to detect equids with specific antibodies for . Molecular detection of by nested PCR was performed on the tissue samples collected. Tissue samples were tested by murine bioassay in an attempt to isolate either the parasite or the parasite DNA. Real-time PCR was performed on the brain samples from 1...
Rar V, Marchenko V, Suntsova O, Epikhina T, Tikunov A, Meltsov I, Fedorets V, Igolkina Y, Kozlova I, Tikunova N.Equine piroplasmosis (EP) is a global worldwide infection, which can lead to the death of animals. Despite the causative agents of EP being well studied, there are no data on the distribution and genetic characteristics of EP agents in any region of Russia. In this study, blood samples from 750 horses from Novosibirsk province, Irkutsk province, and Altai region of Russian Siberia were examined for the presence of EP agents. Theileria equi and Babesia caballi were detected in all examined regions, with mean prevalence rates of 60.4% and 7.2%, respectively. The identified pathogens were genetic...
Grabatin M, Fux R, Zablotski Y, Goehring LS, Witte TS.Contagious equine metritis (CEM) is caused by Taylorella equigenitalis. It is a venereal disease that is detected in some breeds more than others and can cause temporary infertility with substantial costs for regular testing, sanitation and retesting. There was a perceived increase in T. equigenitalis-positive cases in Icelandic intact males where natural cover is common. Objective: We aimed to investigate the prevalence of T. equigenitalis in Icelandic intact males and compare to draught horse and Haflinger intact males. We hypothesised that prevalence of T. equigenitalis is higher in Iceland...
Gonzalez-Obando J, Zuluaga-Cabrera A, Moreno I, Úsuga J, Ciuderis K, Forero JE, Diaz A, Rojas-Arbeláez C, Hernández-Ortiz JP, Ruiz-Saenz J.Equine influenza is a viral disease caused by the equine influenza virus (EIV), and according to the WOAH, it is mandatory to report these infections. In Latin America and Colombia, EIV risk factors have not been analyzed. The objective of this research is to perform an epidemiological and molecular analysis of the EIV in horses with respiratory symptoms from 2020 to 2023 in Colombia. Molecular EIV detection was performed using RT-qPCR and nanopore sequencing. A risk analysis was also performed via the GEE method. A total of 188 equines with EIV respiratory symptoms were recruited. The positiv...
Yi M, Asgenbaatar N, Wang X, Ulaangerel T, Shen Y, Wen X, Du M, Dong X, Dugarjav M, Bou G.DNA methyltransferases (DNMTs) are important epigenetic modification during spermatogenesis. To further evaluate the pattern of DNMTs in horse testes during development, we investigated the expression and localization of DNMT1, DNMT3a and DNMT3b at different time points. The qRT-PCR results showed that DNMT1 expression was maintained in testes tissue from 6-month-old (0.5y) to 2-year-old (2y) of age and decreased after 3-year-old (3y) (P < 0.01). The expression levels of DNMT3a and DNMT3b peaked in testes tissue at 3y (P < 0.01). At 4-year-old (4y), the expression of DNMT3a and DNMT3b was ...
Zu H, Sun R, Li J, Guo X, Wang M, Guo W, Wang X. subspecies () is the causative pathogen of strangles in horses, donkeys, and other equine animals. Strangles has spread globally and causes significant losses to the horse industry. In response to the urgent need for effective disease control, this study introduces a novel nucleic acid diagnostic method known as a real-time recombinase-assisted amplification (RAA) assay, developed based on the gene, for the rapid detection of nucleic acid. The real-time RAA method employs specifically designed probes and primers targeting the gene, enhancing the overall specificity and sensitivity of the ...
Inflammatory myopathy and perivasculitis have been recently described in horses with chronic equine piroplasmosis (EP). These alterations may be linked to poor performances. The aims of this study were to evaluate the prevalence for EP in clinically healthy Italian Standardbred (IS) racehorses and to compare laboratory parameters and performance metrics between positive and negative horses. Real-time PCR was applied for the detection of T. equi and B. caballi positivity. Haematology parameters, blood chemistry results, subjective muscle mass scores, and performance metrics were compared betwee...
Ivanova Z, Petrova V, Grigorova N, Vachkova E.Obtaining accurate and reliable gene expression results in real-time RT-PCR (qRT-PCR) data analysis requires appropriate normalization by carefully selected reference genes, either a single or a combination of multiple housekeeping genes (HKGs). The optimal reference gene/s for normalization should demonstrate stable expression across varying conditions to diminish potential influences on the results. Despite the extensive database available, research data are lacking regarding the most appropriate HKGs for qRT-PCR data analysis in rabbit and horse adipose-derived stem cells (ASCs). Therefore,...
Kinoshita Y, Niwa H, Uchida-Fujii E, Ueno T. is the predominant equine sp. isolated from clinically normal horses and is suspected to be associated with inflammatory airway disease in which cough is the primary sign. Quantitative evaluation of bacterial counts is useful in assessing the association between the bacteria in samples and observed clinical signs, but this evaluation has been difficult with conventional culture methods of given the need for pre-enrichment using liquid cultures. We established a quantitative real-time PCR (qPCR) assay for the quantification of , targeting the hypothetical protein FJM08_00025. We confirmed it...
Lawton K, Runk D, Hankin S, Mendonsa E, Hull D, Barnum S, Pusterla N.The aim of this study was to use environmental sampling to determine the frequency of detection of selected equine respiratory viruses and bacteria in horses attending a multi-week equestrian show during the winter months. At four time points during showing, environmental sponge samples were collected from all stalls on the property and tested for the presence of equine herpesvirus-1 (EHV-1), EHV-2, EHV-4, equine influenza virus (EIV), equine rhinitis B virus (ERBV), Streptococcus equi ss. equi (S. equi), and S. equi ss. zooepidemicus (S. zooepidemicus) using real-time PCR (PCR). Environmental...
Li S, Guo K, Wang X, Lin Y, Wang J, Wang Y, Du C, Hu Z, Wang X.Equine infectious anemia (EIA) has a worldwide distribution and causes severe economic losses to the equine industry. The EIA virus (EIAV) genome sequences from different countries are highly diverse, which poses a great challenge for pathogen identification with PCR. Phylogenetic analysis showed that although gag is the most conserved structural gene, it still has great genome variability. Currently, most existing PCR methods are designed based on the gag gene sequence and therefore do not cover all the viral strains, especially Asian EIAV strains. In this study, we developed a tat-gag-based ...
Dorrego A, Camino E, Gago P, Buendia-Andres A, Acurio K, Gonzalez S, de Juan L, Cruz-Lopez F.Equine piroplasmosis (EP) is caused by Theileria equi and Babesia caballi, transmitted by tick vectors. Horses can suffer an acute, subacute, and chronic forms of the disease, with clinical signs such as poor performance, fever, pale mucosal membranes, and jaundice. The diagnosis of EP subclinical cases is complex due to the sensitivity of real-time PCR and the limited parasite load in some carriers, making it challenging to differentiate them from seropositive, PCR negative (S+PCR-) individuals. This study aimed to describe haematological and biochemical changes in asymptomatic EP carriers, E...
Velloso Alvarez A, Jose-Cunilleras E, Dorrego-Rodriguez A, Santiago-Llorente I, de la Cuesta-Torrado M, Troya-Portillo L, Rivera B, Vitale V....Real-time PCR is the diagnostic technique of choice for the diagnosis and control of equine herpesvirus-1 (EHV-1) in an outbreak setting. The presence of EHV-1 in nasal swabs (NS), whole blood, brain and spinal cord samples has been extensively described; however, there are no reports on the excretion of EHV-1 in urine, its DNA detection patterns, and the role of urine in viral spread during an outbreak. Objective: To determine the presence of EHV-1 DNA in urine during natural infection and to compare the DNA detection patterns of EHV-1 in urine, buffy coat (BC) and NS. Methods: Descriptive st...
Martínez-Sáez L, Dulac Q, Montaner-Angoiti E, Marín-García PJ, Llobat L.Leishmaniosis is a zoonosis caused by spp., an intracellular protozoan parasite. This parasite is transmitted by sandflies, and the disease is endemic in the Mediterranean basin. In recent years, the number of species which could be a reservoir of the parasite is increased. One of the most relevant species is the horse, due to their contact with humans and ability to control the disease, thus being a possible silent reservoir. In this study, we have analyzed the prevalence and factors related to infection in healthy horses in the Mediterranean region. Epidemiological data and serum samples w...
Shakeel M, Yoon M.This study investigated the effect of heat stress on stallion testicular cells (TCs) and the effect of insulin-like growth factor (IGF)-1 on TC viability, proliferation, and apoptosis, including different stages of germ cells. TCs were divided into control or treatment groups with 0.01, 0.1, 1, 10, and 100 ng/mL of recombinant human IGF-1 (rhIGF-1) for 24 h at 34 °C and 37 °C. The population and viability were measured before and after treatment. The effects of rhIGF-1 on TC viability, proliferation, and apoptosis were determined using RT-qPCR. Proliferating cell nuclear antigen (PCNA) a...
Enriquez CK, Morrow JK, Graves A, Johnson A.Equine protozoal myeloencephalitis (EPM) caused by Sarcocystis neurona remains an antemortem diagnostic challenge in some horses. Recent work suggested the use of real-time PCR (rtPCR) on cerebrospinal fluid (CSF) as a promising diagnostic tool. Objective: To evaluate the sensitivity and specificity of S. neurona rtPCR on CSF for EPM diagnosis using horses with EPM and S. neurona-seropositive horses with other neurologic conditions. Methods: Ninety-nine horses with neurologic disease that underwent complete neurologic examination, CSF collection, and, if euthanized, necropsy including the cent...
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...
Balasuriya UB, Leutenegger CM, Topol JB, McCollum WH, Timoney PJ, MacLachlan NJ.A one-tube real-time TaqMan reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of equine arteritis virus (EAV). The test was validated using the seminal plasma and nasal secretions of infected horses that were proven to contain EAV by traditional virus isolation in rabbit kidney thirteen (RK-13) cells, as well as a variety of cell culture-propagated European and North American strains of EAV. The primers and a fluorogenic TaqMan probe were designed to amplify and detect a highly conserved region of open reading frame 7 (ORF7) of EAV. The real-time Ta...
Bhoora R, Quan M, Franssen L, Butler CM, van der Kolk JH, Guthrie AJ, Zweygarth E, Jongejan F, Collins NE.A quantitative real-time polymerase chain reaction (qPCR) assay using a TaqMan minor groove binder (MGB) probe was developed for the detection of Babesia caballi infection in equids from South Africa. Nine previously published sequences of the V4 hypervariable region of the B. caballi 18S rRNA gene were used to design primers and probes to target unique, conserved regions. The B. caballi TaqMan MGB qPCR assay was shown to be efficient and specific. The detection limit, defined as the concentration at which 95% of positive samples can be detected, was determined to be 0.000114% parasitized eryt...
Lavoie JP, Cesarini C, Lavoie-Lamoureux A, Moran K, Lutz S, Picandet V, Jean D, Marcoux M.There is limited information relating bronchoalveolar lavage (BAL) cytology and cytokine messenger ribonucleic acid (mRNA) expression in racehorses with inflammatory airway disease (IAD). HYPOTHESIS AND OBJECTIVE: We hypothesize that cytokine expression in BAL cells would correlate with cytology. Thus, we evaluated the mRNA expression of selected cytokines in BAL cells in racehorses with exercise intolerance and lower airway inflammation. Methods: Thirty-one client-owned Standardbred racehorses with exercise intolerance. Methods: Prospective, observational study. Cells were obtained by BAL, an...
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,...
Allen GP, Breathnach CC.Neurological disease in horses caused by infection with certain 'paralytic' strains of equine herpesvirus-1 (EHV-1) is a potentially devastating condition the pathogenesis of which is poorly understood. Preliminary observations in both experimentally induced and naturally occurring cases of the central nervous system disease have revealed a more robust cell-associated viraemia in horses infected with paralytic isolates of EHV-1, relative to horses infected with abortigenic isolates. To investigate further this pathogenesis-relevant question, the present study was performed using a greater numb...
Klohonatz KM, Cameron AD, Hergenreder JR, da Silveira JC, Belk AD, Veeramachaneni DN, Bouma GJ, Bruemmer JE.During early pregnancy, the conceptus and mare communicate to establish pregnancy. Cell-secreted vesicles (e.g., exosomes) have been reported in serum. Exosomes contain bioactive materials, such as miRNA, that can mediate cell responses. We hypothesized that a) exosomes are present in mare circulation and quantity varies with pregnancy status, b) exosomes contain miRNAs unique to pregnancy status, and c) miRNAs target pathways in endometrium based upon pregnancy status of the mare. First, serum samples were obtained from mares in a crossover design, with each mare providing samples from a preg...
Hussey SB, Clark R, Lunn KF, Breathnach C, Soboll G, Whalley JM, Lunn DP.Equine herpesvirus-1 (EHV-1) infection is common in young horses throughout the world, resulting in respiratory disease, epidemic abortion, sporadic myelitis, or latent infections. To improve on conventional diagnostic tests for EHV-1, a real-time polymerase chain reaction (PCR) technique was developed, using primers and probes specific for the EHV-1 gB gene. Amplification efficiencies of 100% +/- 5% were obtained for DNA isolated from a plasmid, infected peripheral blood mononuclear cells (PBMCs), and nasal secretions from infected ponies. The dynamic range of the assay was 8 log10 dilutions,...
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...
Klein C, Troedsson MH.Establishment and maintenance of pregnancy are critically dependent on embryo-maternal communication during the preimplantation period. The horse is one of the few domestic species in which the conceptus-derived pregnancy recognition signal has not been identified. To gain new insights into the factors released by the equine conceptus, transcriptional profiling analyses of conceptuses retrieved 8, 10, 12, and 14 days after ovulation were performed using a whole-genome microarray. Selected array data were confirmed using quantitative PCR, and the expression of proteins of interest was confirmed...
Szóstek AZ, Lukasik K, Galvão AM, Ferreira-Dias GM, Skarzynski DJ.The aim of the study was to characterize endometrial mRNA transcription, immunolocalization, and protein expression of interleukin (IL) 1alpha, IL1beta, IL6, and IL1RI, IL1RII, and IL6Ralpha/beta in the course of endometrosis during the estrous cycle. Additionally, the influence of IL1alpha, IL1beta, and IL6 on prostaglandin (PG) secretion and PG synthase mRNA transcription in endometrial tissue during endometrosis was investigated. The endometrial samples were obtained at the early (n = 12), mid- (n = 12), and late (n = 12) luteal phases and at the follicular (n = 12) phase of the estrous cyc...
Liburt NR, Adams AA, Betancourt A, Horohov DW, McKeever KH.Studies have demonstrated increases in mRNA expression for inflammatory cytokines following exercise in horses and have suggested those markers of inflammation may play a role in delayed onset muscle soreness. However, measurement of mRNA expression in white blood cells is an indirect method. No studies to date have documented the cytokine response to exercise directly in muscle in horses. Objective: This study tested the hypothesis that exercise increases cytokine markers of inflammation in blood and muscle. Methods: Blood and muscle biopsies were obtained from 4 healthy, unfit Standardbred m...
Finley MR, Li Y, Hua F, Lillich J, Mitchell KE, Ganta S, Gilmour RF, Freeman LC.In dogs and in humans, potassium channels formed by ether-a-go-go-related gene 1 protein ERG1 (KCNH2) and KCNQ1 alpha-subunits, in association with KCNE beta-subunits, play a role in normal repolarization and may contribute to abnormal repolarization associated with long QT syndrome (LQTS). The molecular basis of repolarization in horse heart is unknown, although horses exhibit common cardiac arrhythmias and may receive drugs that induce LQTS. In horse heart, we have used immunoblotting and immunostaining to demonstrate the expression of ERG1, KCNQ1, KCNE1, and KCNE3 proteins and RT-PCR to det...
Cook RF, Cook SJ, Li FL, Montelaro RC, Issel CJ.A single-tube reverse transcriptase-polymerase chain reaction (RT-PCR) using a fluorogenic real-time PCR detection method is described for the quantitation of equine infectious anemia virus (EIAV) RNA in the plasma of equids. To compensate for variations inherent in sample preparation a multiplex real-time RT-PCR system was developed that permitted the simultaneous calculation of the nucleic acid recovery rate along with the copy number of viral RNA molecules. Detection of EIAV RNA was linear from 10(9) to 10(1) molecules with intra- and inter-assay variability of less than 1% at 10(8), 10(6),...
Nazari F, Parham A, Maleki AF.Quantitative real time reverse transcription PCR (qRT-PCR) is one of the most important techniques for gene-expression analysis in molecular based studies. Selecting a proper internal control gene for normalizing data is a crucial step in gene expression analysis via this method. The expression levels of reference genes should be remained constant among cells in different tissues. However, it seems that the location of cells in different tissues might influence their expression. The purpose of this study was to determine whether the source of mesenchymal stem cells (MSCs) has any effect on exp...
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...
Diallo IS, Hewitson G, Wright LL, Kelly MA, Rodwell BJ, Corney BG.A multiplex real-time PCR was designed to detect and differentiate equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). The PCR targets the glycoprotein B gene of EHV-1 and EHV-4. Primers and probes were specific to each equine herpesvirus type and can be used in monoplex or multiplex PCRs, allowing the differentiation of these two closely related members of the Alphaherpesvirinae. The two probes were minor-groove binding probes (MGB) labelled with 6-carboxy-fluorescein (FAM) and VIC for detection of EHV-1 and EHV-4, respectively. Ten EHV-1 isolates, six EHV-1 positive clinical samples...
Pusterla N, Wilson WD, Mapes S, Finno C, Isbell D, Arthur RM, Ferraro GL.The objective of this study was to determine viral loads, strain (neuropathogenic versus non-neuropathogenic) and state (lytic, non-replicating, latent) of equine herpesvirus-1 (EHV-1) by real-time polymerase chain reaction (PCR) in the blood and nasopharyngeal secretions of adult horses following natural exposure. The index case, a 4-year-old Thoroughbred gelding with confirmed EHV-1 myeloencephalopathy, as well as potentially exposed horses, were sampled over a period of 3 weeks. The study population comprised of 39 adult Thoroughbred horses and 35 adult "pony" and outrider horses of various...
Buling A, Criado-Fornelio A, Asenzo G, Benitez D, Barba-Carretero JC, Florin-Christensen M.The haemoparasites Babesia bovis and Babesia bigemina affect cattle over vast areas of the tropics and temperate parts of the world. Microscopic examination of blood smears allows the detection of clinical cases of babesiosis, but this procedure lacks sensitivity when parasitaemia levels are low. In addition, differentiating between similar haemoparasites can be very difficult. Molecular diagnostic procedures can, however, overcome these problems. This paper reports a quantitative PCR (qPCR) assay involving the use of SYBR Green. Based on the amplification of a small fragment of the cytochrome...
Huang H, Lavoie-Lamoureux A, Moran K, Lavoie JP.Little is known concerning the possible contribution of T helper 2 (Th2)-type cytokines to the recruitment of neutrophils into the lung tissue. In the present study, endothelial cells from equine pulmonary arteries were cultured in the presence of recombinant equine (re) IL-4 and reIL-5, and the cytokine mRNA expression of molecules implicated in the chemotaxis and migration of neutrophils was studied using real-time RT-PCR. The functional response of reIL-4-induced endothelial cell stimulation on neutrophil migration was also studied using a chemotaxis chamber. ReIL-4 either increased the exp...
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...
McGivney BA, Browne JA, Fonseca RG, Katz LM, Machugh DE, Whiston R, Hill EW.Myostatin, encoded by the MSTN gene, is a member of the TGF-β superfamily that regulates skeletal muscle development. A MSTN SNP significantly associated with Thoroughbred horse racing phenotypes has recently been identified as well as significant reductions in Thoroughbred skeletal muscle gene expression for three transcripts 400-1500 base pairs downstream of the MSTN gene following a period of training. Together, these findings indicate that MSTN genotypes may influence MSTN gene expression. To investigate this, MSTN mRNA expression was measured in biopsies from the middle gluteal muscle fr...
Isgren CM, Edwards T, Pinchbeck GL, Winward E, Adams ER, Norton P, Timofte D, Maddox TW, Clegg PD, Williams NJ.This study investigated changes over time in the epidemiology of extended-spectrum β-lactamase (ESBL) producing Escherichia coli within a single equine referral hospital in the UK. Faecal samples were collected from hospitalised horses in 2008 and 2017, processed using selective media and standard susceptibility laboratory methods. A novel real-time PCR with high resolution melt analysis was used to distinguish bla and bla within CTX-M-1 group. Results: In 2008, 457 faecal samples from 103 horses were collected, with ESBL-producing E. coli identified in 131 samples (28.7, 95% CI 24.6-33.1). I...
Balasuriya UB, Lee PY, Tiwari A, Skillman A, Nam B, Chambers TM, Tsai YL, Ma LJ, Yang PC, Chang HF, Wang HT.Equine influenza (EI) is an acute, highly contagious viral respiratory disease of equids. Currently, equine influenza virus (EIV) subtype H3N8 continues to be the most important respiratory pathogen of horses in many countries around the world. The need to achieve a rapid diagnosis and to implement effective quarantine and movement restrictions is critical in controlling the spread of EIV. In this study, a novel, inexpensive and user-friendly assay based on an insulated isothermal RT-PCR (iiRT-PCR) method on the POCKIT™, a field-deployable device, was described and validated for point-of-nee...
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...
Spier SJ, Leutenegger CM, Carroll SP, Loye JE, Pusterla JB, Carpenter TE, Mihalyi JE, Madigan JE.To develop and use a sensitive molecular assay for detecting the phospholipase D (PLD) exotoxin gene of Corynebacterium pseudotuberculosis in an attempt to identify insect vectors that may be important in transmission of clinical disease in horses. Methods: 2,621 flies of various species. Methods: A real-time polymerase chain reaction (PCR)-based fluorogenic 5' nuclease (TaqMan) system (ie, TaqMan PCR assay) was developed for the detection of the PLD gene in insects. Flies were collected monthly (May to November 2002) from 5 farms in northern California where C. pseudotuberculosis infection in...
Pusterla N, Magdesian KG, Mapes S, Leutenegger CM.To determine gene expression of selected molecular markers (tumor necrosis factor [TNF]-alpha, interleukin [IL]-1beta, IL-6, IL-8, IL-10, procalcitonin [PCT], and transforming growth factor [TGF]-beta) in the blood of healthy and sick foals. Methods: 28 sick foals without sepsis, 21 foals with sepsis, and 21 healthy foals. Methods: Total RNA was extracted from blood samples and converted into complementary DNA (cDNA). Gene expression was measured for the molecular markers by use of real-time PCR assay, and final quantitation was performed with the comparative threshold cycle method. Results: S...
Lindahl S, Båverud V, Egenvall A, Aspán A, Pringle J.Strangles is a contagious equine-specific disease caused by Streptococcus equi subsp. equi. Unfortunately, detection of S. equi can fail in up to 40% of horses with strangles. Whereas recent molecular biologic methods and sampling techniques have improved recovery of S. equi optimal sampling methods and laboratory analyses remain ill-defined. Objective: To determine the yield of S. equi from horses with acute strangles in confirmed outbreaks by field-sampling methods subjected to culture and biochemical identification, and real-time PCR directly and after culture. Methods: Fifty-seven horses o...
Vázquez-Morón S, Avellón A, Echevarría JE.The Lyssavirus genus includes seven species or genotypes named 1-7. Rabies genotypes correlate with geographical distribution and specific hosts. Co-circulation of different lyssaviruses, imported cases, and the presence of unknown viruses, such as Aravan, Khujand, Irkut and West Caucasian Bat Virus, make it necessary to use generic methods able to detect all lyssaviruses. Primer sequences were chosen from conserved regions in all genotypes in order to optimise a generic RT-PCR. Serial dilutions of 12 RNA extracts from all seven Lyssavirus genotypes were examined to compare the sensitivity of ...
Leise BS, Faleiros RR, Watts M, Johnson PJ, Black SJ, Belknap JK.There is a need to assess the laminar inflammatory response in a laminitis model that more closely resembles clinical cases of sepsis-related laminitis than the black walnut extract (BWE) model. Objective: To determine if a similar pattern of laminar inflammation, characterised by proinflammatory cytokine expression, occurs in the CHO model of laminitis as has been previously reported for the BWE model. Methods: Sixteen horses administered 17.6 g of starch (85% corn starch/15% wood flour)/kg bwt via nasogastric (NG) tube were anaesthetised either after developing a temperature>38.9°C (DEV ...
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...
