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Topic:Polymerase Chain Reaction
Polymerase Chain Reaction (PCR) is a molecular biology technique used to amplify specific DNA sequences, allowing for detailed genetic analysis in horses. This method enables the detection and quantification of genetic material, facilitating research in areas such as genetic disorders, infectious diseases, and population genetics in equine species. PCR applications in horses include identifying pathogens, verifying parentage, and studying genetic variations. The technique's sensitivity and specificity make it a valuable tool in equine veterinary diagnostics and research. This page compiles peer-reviewed research studies and scholarly articles that explore the applications, methodologies, and advancements of PCR in equine science.
Construction of a recombinant plasmid as reaction control in routine PCR for detection of contagious equine metritis (CEM-PCR). Contagious equine metritis (CEM) is a highly contagious bacterial venereal disease of horses caused by Taylorella equigenitalis. CEM-PCR is a semi-nested PCR method for detecting this bacterium. Although this technique is regarded as a sensitive diagnostic method for CEM, there are risks of it generating false positive and false negative results. In this study, we constructed a recombinant plasmid (CEM-POS) as reaction control to assure adequate PCR reaction and prevent false positive results caused by contamination of the reaction control in routine CEM-PCR examinations. CEM-POS was construct...
Effects of in vitro exposure to hay dust on expression of interleukin-17, -23, -8, and -1beta and chemokine (C-X-C motif) ligand 2 by pulmonary mononuclear cells isolated from horses chronically affected with recurrent airway disease. To examine effects of in vitro exposure to solutions of hay dust, lipopolysaccharide (LPS), or beta-glucan on cytokine expression in pulmonary mononuclear cells isolated from healthy horses and horses with recurrent airway obstruction (RAO). Methods: 8 RAO-affected and 7 control horses (experiment 1) and 6 of the RAO-affected and 5 of the control horses (experiment 2). Methods: Bronchoalveolar lavage cells were isolated from horses that had been stabled and fed dusty hay for 14 days. Pulmonary mononuclear cells were incubated for 24 (experiment 1) or 6 (experiment 2) hours with PBS solution or...
Equine multinodular pulmonary fibrosis: a newly recognized herpesvirus-associated fibrotic lung disease. Pulmonary fibrosis and interstitial lung disease are poorly understood in horses; the causes of such conditions are rarely identified. Equine herpesvirus 5 (EHV-5) is a gamma-herpesvirus of horses that has not been associated with disease in horses. Pathologic and virologic findings from 24 horses with progressive nodular fibrotic lung disease associated with EHV-5 infection are described and compared with 23 age-matched control animals. Gross lesions consisted of multiple nodules of fibrosis throughout the lungs. Histologically, there was marked interstitial fibrosis, often with preservation ...
A soluble secreted glycoprotein (eCLCA1) is overexpressed due to goblet cell hyperplasia and metaplasia in horses with recurrent airway obstruction. The equine putative chloride channel protein eCLCA1 is thought to be critically involved in the pathogenesis of recurrent airway obstruction (RAO) via modulation of the hydration of airway mucins. A recent study revealed a strong increase of eCLCA1 messenger ribonucleic acid (mRNA) in the lungs of horses with RAO. In this study, eCLCA1 protein and mRNA expression were quantified in airway goblet cells of 9 horses affected with RAO and 9 control horses by using immunohistochemistry and laser microdissection followed by real-time quantitative reverse transcription polymerase chain reaction, resp...
Characterization of viral loads, strain and state of equine herpesvirus-1 using real-time PCR in horses following natural exposure at a racetrack in California. 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...
Repeated high dose imidocarb dipropionate treatment did not eliminate Babesia caballi from naturally infected horses as determined by PCR-reverse line blot hybridization. Imidocarb treatment of horses infected with Babesia caballi is supposed to eliminate the infection, but data on the efficacy of this treatment is scarce. The study presented here concerns four Paso Fino horses, which were imported into the island of Curacao on the basis of a piroplasmosis negative complement fixation test (CFT). Upon re-testing with an indirect fluorescent antibody test immediately after arrival in Curacao, two horses appeared to have antibodies to B. caballi and all horses had antibodies to Theileria equi. Subsequent testing with polymerase chain reaction combined with a reve...
Diagnostic real-time PCR assay for the quantitative detection of Theileria equi from equine blood samples. We developed a TaqMan real-time polymerase chain reaction (PCR) assay for the quantitative detection of Theileria equi from the in vitro-cultured parasite and field blood samples collected from horses living in Ghana and Brazil. The detection limit for the assay was determined to be 1.5 parasites/microl per sample, and the quantitative capacity was demonstrated using the in vitro-cultured parasite. For field applications, the real-time PCR assay was compared to a previously established nested PCR assay used as the gold standard for the real-time PCR assay. Of 65 field blood samples, 46 samples...
Diagnosis of equine infectious anaemia during the 2006 outbreak in Ireland. In 2006 there was an outbreak of equine infectious anaemia (EIA) in Ireland. This paper describes the use of the diagnosis of clinical and subclinical cases of the disease. In acute cases the ELISAs and the immunoblot were more sensitive than the AGID. In one mare, fluctuating antibody levels were observed in all the serological assays before it seroconverted by AGID. Viral RNA and DNA were detected by RT-PCR and PCR in all the tissues from the infected animals examined postmortem. The PCR detected viral DNA in plasma regardless of the stage of the disease. In contrast, the RT-PCR detected RNA...
Comparison of polymerase chain reaction methods for the detection of Theileria equi infection using whole blood compared with pre-extracted DNA samples as PCR templates. Rapid, efficient, and reproducible procedures for isolating DNA before PCR gene amplification are essential for the diagnosis of piroplasms. In this study, we evaluated the ease and reliability of detecting Theileria equi by PCR using pre-extracted DNA samples (by QIAamp DNA Mini Kit and phenol-chloroform methods) compared with blood spotted on FTA cards as PCR templates. Although minimal variations in limit of detection were observed among the methods compared, overall, the use of pre-extracted DNA samples and blood spotted on FTA cards had comparable detection limits. These results indicate ...
Passive transfer of Theileria equi antibodies to neonate foals of immune tolerant mares. Equine babesiosis, a tick transmitted haemoprotozoan disease caused by Theileria equi is globally distributed and responsible for heavy economic losses to the equine husbandry. Equids reared in endemic areas usually pick up infection at an early age and become immune tolerant throughout their life span. We studied the level of passively transferred antibodies in neonate foals born from pre-immuned mares. Latently T. equi infected pre-immuned pony and donkey mares (three each) were selected and T. equi antibody titres in neonates was monitored till 90 days post foaling (DPF) by applying Dot-ELI...
Detection of Helicobacter-like DNA in the gastric mucosa of Thoroughbred horses. To assess the presence of Helicobacter DNA in the gastric mucosa Thoroughbred horses. Results: Squamous and glandular mucosa samples were collected from 20 Thoroughbreds. None of these horses had shown any clinical symptoms of gastrointestinal disease. Necropsy tissues were analysed using histopathological techniques and a Helicobacter genus-specific PCR assay followed by sequencing of the amplicons. Seven horses were diagnosed with gastric ulceration, five with gastritis and six with both pathologies. Only two horses had a healthy gastric mucosa. Helicobacter-like DNA was detected in two out ...
Molecular diagnosis of equid summer sores. Equine cutaneous habronemosis, also known as "summer sores", is a parasitic infection caused by larvae of Habronema microstoma and Habronema muscae (Nematoda, Spirurida) released by dung-inhabiting fly vectors on abraded skin, skin wounds or muco-cutaneous transition sites. Larvae induce a local inflammatory reaction characterised by itching, granulomatous, ulcerated and, often non-healing, lesions. The diagnosis of summer sores may be unreliable mainly because of the limits of clinical and microscopic examination. The applicability of a semi-nested PCR assay developed for the diagnosis of gas...
Comparison of different molecular methods for assessment of equine arteritis virus (EAV) infection: a novel one-step MGB real-time RT-PCR assay, PCR-ELISA and classical RT-PCR for detection of highly diverse sequences of Slovenian EAV variants. In the present study, a new one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) strategy with minor-groove-binder (MGB) technology for the detection of EAV from 40 semen samples of Slovenian carrier stallions was tested. A novel MGB probe (EAVMGBpr) and a reverse primer (EAV-R) based on the multiple sequence alignment of 49 different EAV strain sequences of the highly conserved ORF7 (nucleocapsid gene) were designed. The performance of the assay was compared with different molecular detection methods. Three different primer pairs targeting the ORF1b and ORF7 were used, ...
Detection and molecular characterization of Babesia caballi and Theileria equi isolates from endemic areas of Brazil. Blood samples were collected from 487 adult horses, including 83 pregnant mares, at a slaughterhouse located in Araguari, Minas Gerais State, Brazil. For each blood sample, the packed cell volume (PCV) was determined, and Giemsa-stained smears were microscopically examined for the presence of hemoparasites. The plasma was examined by the indirect fluorescent antibody test for detection of antibodies against Babesia caballi and Theileria equi. In addition, DNA was extracted and analyzed by a multiplex real-time polymerase chain reaction (PCR), specific for B. caballi and T. equi. Products of PC...
Validation of quantitative polymerase chain reaction assays for measuring cytokine expression in equine macrophages. The study of the equine immune system and inflammatory responses, by measuring cytokine expression, can provide important insight into disease pathogenesis in the horse. A set of quantitative real-time polymerase chain reaction (QPCR) assays for the equine cytokines IL-1alpha, IL-1beta, IL-6, IL-8 and TNF-alpha were validated using QPCR primers and probes which were generated for the equine IL-1alpha, IL-1beta, IL-6, IL-8, TNF-alpha and 18S genes. Amplification efficiency, intra-assay and inter-assay variation were determined using 10-fold dilutions of plasmid for each gene. Under these condit...
Gag genetic heterogeneity of equine infectious anemia virus (EIAV) in naturally infected horses in Canada. Gag genetic heterogeneity of equine infectious anemia virus (EIAV) variants in naturally infected horses in Canada was studied since very limited information is available on the variability of EIAV Gag sequences in public database. A phylogenetic analysis based on 414nts of Gag gene sequences amplified by a nested polymerase chain reaction (PCR) revealed the distinct divergence of these variants compared to other published strains in a corresponding region. Significant predicted amino acid sequence variations were also identified in an immunorelevant region within this fragment which correspon...
Molecular profiling of Lactobacillus, Streptococcus, and Bifidobacterium species in feces of active racehorses. Diversity and compositions of the Lactobacillus, Streptococcus, and Bifidobacterium group in the feces of six healthy, actively racing horses (Thoroughbreds) were analyzed by using PCR-denaturing gradient gel electrophoresis (DGGE) and real-time PCR with primer sets specific for each group. PCR-DGGE analysis of the feces showed that Lactobacillus equi, Lactobacillus johnsonii, a phylogenetic relative of Lactobacillus salivarius, a phylogenetic relative of Lactobacillus gastricus, and Weissella confusa were predominant in almost all of the feces tested, and Streptococcus bovis/Streptococcus equ...
Acute in vivo interactions of Helicobacter equorum with its equine host. A novel urease-negative Helicobacter species has been isolated from faecal samples of clinically healthy horses, but no information is available about the main sites of colonisation in the equine gastrointestinal tract nor is the pathogenic potential of this microorganism known. An experimental infection in horses was therefore carried out. Methods: Four horses were infected with H. equorum strain CCUG 52199T and subjected to euthanasia at 10 (n = 2) and 30 days (n = 2) post inoculation. A fifth animal was inoculated with phosphate buffered saline and used as control. Gastrointestinal samples ...
Seasonal relationships between dopamine D1 and D2 receptor and equine FSH receptor mRNA in equine ovarian epithelium. Dopamine (DA) blockade during anestrus or early spring transition can facilitate ovarian recrudescence and advance the timing of the first ovulation of the season. Some laboratories have reported variable results using DA antagonists to stimulate follicular growth during the mid-portion of the anestrual period. Differences in DA antagonist efficacy may be due to the FSH secretory status of the anestrous mare and the presence or absence of functional ovarian FSH receptors. We hypothesize that direct ovarian dopaminergic input can affect follicular growth through regulation of FSH receptor (FSHr...
Prevalence of equine herpesvirus-1 infection among Thoroughbreds residing on a farm on which the virus was endemic. To determine the incidence of equine herpesvirus-1 (EHV-1) infection among Thoroughbreds residing on a farm on which the virus was known to be endemic. Methods: Prospective cohort study. Methods: 10 nonpregnant mares, 8 stallions, 16 weanlings, 11 racehorses, and 30 pregnant mares and their foals born during the 2006 foaling season. Methods: Blood and nasopharygeal swab samples were collected every 3 to 5 weeks for 9 months, and placenta and colostrum samples were collected at foaling. All samples were submitted for testing for EHV-1 DNA with a PCR assay. A type-specific EHV-1 ELISA was used t...
Molecular typing of Sarcocystis neurona: current status and future trends. Sarcocystis neurona is an important protozoal pathogen because it causes the serious neurological disease equine protozoal myeloencephalitis (EPM). The capacity of this organism to cause a wide spectrum of neurological signs in horses and the broad geographic distribution of observed cases in the Americas drive the need for sensitive, reliable and rapid typing methods to characterize strains. Various molecular methods have been developed and used to diagnose EPM due to S. neurona, to identify S. neurona isolates and to determine the heterogeneity and evolutionary relatedness within this specie...
Detection and semi-quantification of Strongylus vulgaris DNA in equine faeces by real-time quantitative PCR. Strongylus vulgaris is an important strongyle nematode with high pathogenic potential infecting horses world-wide. Several decades of intensive anthelmintic use has virtually eliminated clinical disease caused by S. vulgaris, but has also caused high levels of anthelmintic resistance in equine small strongyle (cyathostomin) nematodes. Recommendations aimed at limiting the development of anthelmintic resistance by reducing treatment intensity raises a simultaneous demand for reliable and accurate diagnostic tools for detecting important parasitic pathogens. Presently, the only means available t...
Matrix-encapsulation cell-seeding technique to prevent cell detachment during arthroscopic implantation of matrix-induced autologous chondrocytes. The goal of this study is to evaluate the efficiency of obtaining a large number of viable cells within a construct that will not be detached by high fluid flow during arthroscopic implantation. Methods: Arthroscopic osteochondral biopsy specimens were obtained from the medial femoral trochlea of 8 horses. Chondrocytes were isolated by collagenase digestion and expanded in M199 media until confluency. After 10 to 12 days, cultures were trypsinized and cells resuspended in culture media. Then, 5 x 10(6) cells x mL(-1) were seeded on a culture dish and the same amount in a flask. Once extracellu...
Detection of equine herpesviruses in aborted foetuses by consensus PCR. The major role of EHV-1 in equine abortion is widely reported in the literature but the contribution of EHV-2, EHV-3, EHV-4 or EHV-5 remains less well documented. The objective of this study is to evaluate the contribution of these five different EHVs to equine abortion in a variety of biological tissues using a consensus polymerase chain reaction (PCR). The test was validated for specificity and sensitivity in horses before screening specimens from 407 foetuses, stillbirths and premature foals collected over a 2.5-year interval. Positive results obtained with this assay were compared to other...
[Detection of Babesia caballi (Nuttall, 1910) and Theileria equi (Syn. Babesia equi , Laveran, 1901) by the polymerase chain reaction (PCR) in show and sport horses in the region of Ankara]. The aim of this study was to compare the diagnosis of Babesia caballi and Theileria equi by the polymerase chain reaction (PCR) and microscopic examination of blood specimens collected from show and sport horses in the region of Ankara in 2004. The blood specimens were collected from randomly selected 200 show and sport horses in the region of Ankara during the tick season as well as before and after the tick season for PCR testing. At the same time, Giemsa stained peripheral blood smears were examined for the presence of Babesia spp. and also the horses were examined for the presence of ticks...
Genetic modification of chondrocytes with insulin-like growth factor-1 enhances cartilage healing in an equine model. Gene therapy with insulin-like growth factor-1 (IGF-1) increases matrix production and enhances chondrocyte proliferation and survival in vitro. The purpose of this study was to determine whether arthroscopically-grafted chondrocytes genetically modified by an adenovirus vector encoding equine IGF-1 (AdIGF-1) would have a beneficial effect on cartilage healing in an equine femoropatellar joint model. A total of 16 horses underwent arthroscopic repair of a single 15 mm cartilage defect in each femoropatellar joint. One joint received 2 x 10(7) AdIGF-1 modified chondrocytes and the contralateral...
Recurrent uveitis in horses: vitreal examinations with ultrastructural detection of leptospires. This study documents the examination of 17 horses (both sexes, 3-18 years old) suffering from spontaneous equine recurrent uveitis (ERU). Vitreal samples obtained by pars plana vitrectomy were examined macroscopically and ultrastructurally, and in most cases also by cultural examination, by microscopic agglutination test (MAT) and by polymerase chain reaction. In 24% (4/17) of the animals, ultrastructural examination by electron microscopy revealed intact leptospiral bacteria in the vitreous. The leptospires were detected freely in the vitreous and also incorporated by a phagocyte. They were s...
Real-time quantitative RT-PCR and PCR assays for a novel European field isolate of equine infectious anaemia virus based on sequence determination of the gag gene. In 2006, an outbreak of equine infectious anaemia (EIA) occurred in Ireland. The initial source of the outbreak is believed to have been contaminated plasma imported from Italy. This paper presents the nucleotide sequence of the gag gene of the virus identified in Ireland (EIAV(Ire)), the first for a European strain of EIAV. Comparison of the gag gene with North American and Asian strains of the virus showed that the gag gene is less well conserved than previously believed, and that EIAV strains can have similar phenotypes despite considerable variations in genotype. On the basis of the deduce...
Genetic relationships of five Indian horse breeds using microsatellite markers. The genetic relationships of five Indian horse breeds, namely Marwari, Spiti, Bhutia, Manipuri and Zanskari were studied using microsatellite markers. The DNA samples of 189 horses of these breeds were amplified by polymerase chain reaction using 25 microsatellite loci. The total number of alleles varied from five to 10 with a mean heterozygosity of 0.58 ± 0.05. Spiti and Zansakari were the most closely related breeds, whereas, Marwari and Manipuri were most distant apart with Nei's DA genetic distance of 0.071 and 0.186, respectively. In a Nei's DA genetic distances based neighbour joining...
Infections caused by pathogenic free-living amebas (Balamuthia mandrillaris and Acanthamoeba sp.) in horses. This article describes amebic infections in 4 horses: granulomatous amebic encephalitis caused by Balamuthia mandrillaris and Acanthamoeba culbertsoni and systemic infections caused by Acanthamoeba sp. The former infection occurred in 1 of 4 horses spontaneously without any underlying conditions; the latter amebic infection was perhaps "opportunistic" considering the visceral involvement by this protozoan in association with Aspergillus sp. and/or Escherichia coli and Pseudomonas sp. The clinicopathologic findings and demonstration of the amebic organisms using immunohistochemical techniques, ...
