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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.
Quantitative real-time PCR for equine cytokine mRNA in nondecalcified bone tissue embedded in methyl methacrylate. Specific amplification and quantitation of nucleic acid sequences by the polymerase chain reaction (PCR) has been extensively used for the detection of viral infection and gene expression. Although successful amplification of DNA and RNA sequences extracted from paraffin embedded tissue have been described, there are presently no reports available regarding RNA analysis from bone and calcified tissues embedded in hydrophobic acrylic resin. Here we describe a general method for quantitation of specific mRNA sequences extracted from undecalcified bone sections, fixed in paraformaldehyde, and emb...
Experimental cross-infections with Ehrlichia phagocytophila and human granulocytic ehrlichia-like agent in cows and horses. Four cows and four horses were infected experimentally with Ehrlichia phagocytophila, the cause of tickborne fever in ruminants, and with human granulocytic ehrlichia-like agent, a recently discovered species that infects people, horses and dogs in the USA and Europe. They were infected in either order, 30 days apart, to investigate serological cross-reactivity within the Ephagocytophila genogroup. The course of infection was assessed by routine clinical, haematological, serological and polymerase chain reaction (PCR) examinations. Two of the cows infected with Ephagocytophila and two of the h...
Salmonella Abortusequi strains of equine origin harbor a 95kb plasmid responsible for virulence in mice. Most Salmonella choleraesuis subsp. choleraesuis serovar Abortusequi strains of equine origin harbor a 95kb plasmid, pSA95. Results of PCR and Southern blot analysis suggest that pSA95 contains spv genes. A pSA95-cured strain of S. Abortusequi was 48 times less virulent to mice than its parental strain. Virulence was restored by reintroduction of pSA95. These results provide clear evidence that pSA95 confers virulence on S. Abortusequi in mice. This is the first report describing a virulence plasmid of S. Abortusequi.
[Cowpox viruses in Germany: an analysis of 5 cases in 1998]. Five case reports on cowpox virus infections in cats, humans, and for the first time in a horse are presented. It becomes obvious that in most cases the diagnosis cowpox is suspected rather late, although fast and reliable diagnostic tools such as pathohistological examination and polymerase chain reaction are available. The threat of a zoonotic transmission mainly through cats is gaining importance. Although wild rodents have been claimed to be the reservoir and source for cowpox viruses in cats, very little is known about the epidemiology of cowpox virus. Based on the different genome organi...
Lawsonia intracellularis-like organism infection in a miniature foal. A 7-month-old foal was admitted to the hospital with a history of lethargy, weight loss, mild diarrhea, and anorexia. A diagnosis of proliferative enteritis caused by Lawsonia intracellularis-like organisms was made after necropsy and histologic examination of the small intestine. Although infection with L intracellularis-like organisms is a rare cause of enteritis in foals, it should be considered in the differential diagnosis, especially if the foal was housed in the proximity of pigs or pig feces. Antemortem diagnosis remains challenging because isolation of the organism in fecal material r...
Detection of new DNA polymerase genes of known and potentially novel herpesviruses by PCR with degenerate and deoxyinosine-substituted primers. A consensus primer PCR approach was used to (i) investigate the presence of herpesviruses in wild and zoo equids (zebra, wild ass, tapir) and to (ii) study the genetic relationship of the herpesvirus of pigeons (columbid herpesvirus 1) to other herpesvirus species. The PCR assay, based on degenerate primers targeting highly conserved regions of the DNA polymerase gene of herpesviruses, was modified by using a mixture of degenerate and deoxyinosine-substituted primers. The applicability of the modification was validated by amplification of published DNA polymerase genes of 16 herpesvirus specie...
Species-specific amplification by PCR of ribosomal DNA from some equine strongyles. The first and second internal transcribed spacer sequences of 28 morphologically-defined species of horse strongyle were characterized, and specific oligonucleotide primers were designed for some species based on the nucleotide differences. Utilizing these primers, a PCR approach was developed for the specific amplification of ribosomal DNA of Strongylus vulgaris, Cyathostomum catinatum, Cylicocyclus nassatus, Cylicostephanus longibursatus or Cylicostephanus goldi. The method allowed the species-specific amplification of parasite DNA derived from faecal samples and/or copro-cultures, demonstra...
Molecular detection of Babesia equi and Babesia caballi in horse blood by PCR amplification of part of the 16S rRNA gene. Babesia equi and Babesia caballi are tick-borne haemoparasites that may cause babesiosis of Equidae. In southern Europe B. equi is enzootic and infections may occur asymptomatically and more frequently than those due to B. caballi. Complement fixation test (CFT) is the official serological test for the diagnosis of equine babesiosis, but it has low sensitivity during early and latent stages of the disease. With the aim of developing more sensitive and rapid direct diagnostic alternatives, PCR systems that amplified DNA targets of 664 or 659 bp regions of the 16S rRNA genes were designed and de...
A PCR based method for the identification of equine influenza virus from clinical samples. In this paper we describe the development of a nested RT-PCR assay for the rapid diagnosis and characterisation of influenza virus directly from clinical specimens. Viral RNA is extracted from nasal swabs by the guanidine thiocyanate extraction method, and subsequently reverse transcribed. The complementary DNA is then used as template in a nested PCR reaction. Primers designed for use in this assay are specific for three templates; (1) the nucleoprotein (NP) gene, (2) the haemagglutinin gene of the H7N7 equine influenza virus (A1), and (3) the haemagglutinin gene of the H3N8 equine influenza ...
Detection of equine arteritis virus in semen by reverse transcriptase polymerase chain reaction-ELISA. The reverse transcriptase polymerase chain reaction (RT-PCR) assay was used to detect Equine Arteritis Virus (EAV) in the semen of 88 horses and 2 donkeys, with neutralising antibodies against EAV, on the basis of the amplification of a 279 bp long fragment located in the viral polymerase gene. The RT-PCR assay revealed the virus at 4 TCID50/ml in cell culture and showed a greater sensitivity (54.4%) than cell culture isolation (33.3%). Moreover, the two samples of donkey semen were found positive. The cDNAs obtained from 14 samples of horse and 2 of donkey semen were sequenced. Comparing the ...
Detection of equine herpesvirus types 2 and 5 (EHV-2 and EHV-5) in Przewalski’s wild horses. In blood samples of seven captive equid species from four German zoos EHV-1 specific antibodies were detected in 76% and EHV-4 specific antibodies in 73% of the 55 animals, whereas 93% were tested positive for EHV-2 and EHV-5, respectively. In only one blood sample from a Przewalski's wild horse EHV-4 DNA was amplified by PCR. From seven Przewalski's wild horses EHV-2, and from another one EHV-5 was isolated by cocultivation. The identity of the virus isolates was verified by PCR and restriction enzyme digestion.
Rapid and sensitive detection of equine arteritis virus in semen and tissue samples by reverse transcription-polymerase chain reaction, dot blot hybridisation and nested polymerase chain reaction. A reverse transcription-polymerase chain reaction (RT-PCR) assay using four different primer pairs for the detection of equine arteritis virus (EAV) RNA in semen and tissue samples was evaluated. A fragment encoding the leader sequence of the EAV genome was most successfully amplified. The specificity and sensitivity of RT-PCR was assessed by virus isolation in cell culture, restriction analysis, dot blot hybridisation and nested PCR. To this end, 23 semen samples from seropositive stallions and 11 tissue samples from 4 aborted foals were tested. Compared to the virus isolation test in cell cu...
The cDNA sequences of equine antioxidative enzyme genes Cu/Zn-SOD and Mn-SOD, and these expressions in equine tissues. The entire cDNA sequences were determined by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques for equine copper/zinc superoxide dismutase (Cu/Zn-SOD) and manganese superoxide dismutase (Mn-SOD) through the use of total RNA extracted from the testis of an adult Thoroughbred. The results revealed a protein coding region for equine Cu/Zn-SOD with bases totaling 465 bp, accompanied by an estimated 154 residues of amino acids. As for equine Mn-SOD, its coding region contained a total of 669 bp and an estimated 222 residues of amino acid...
Serologic testing of horses for granulocytic ehrlichiosis, using indirect fluorescent antibody staining and immunoblot analysis. To diagnose granulocytic ehrlichiosis in horses, compare results of indirect fluorescent antibody (IFA) staining procedures with those of immunoblot analysis, and compare serologic test findings with polymerase chain reaction (PCR) results. Methods: 69 horses with high rectal temperatures (> or = 39 C) and lethargy, anorexia, or limb edema. Methods: 43 convalescent serum samples obtained from 38 horses 2 to 18 weeks after onset of illness were analyzed by use of immunoblot procedures and IFA staining methods, using the NCH-1 or BDS ehrlichial strains. Blood samples from 69 acutely ill horse...
Construction of chromosome-specific paints for meta- and submetacentric autosomes and the sex chromosomes in the horse and their use to detect homologous chromosomal segments in the donkey. A pilot study comparing horse and donkey karyotypes on a molecular basis was initiated using the chromosomal microdissection approach. All equine meta- and submetacentric chromosomes, viz. ECA1 to ECA13 and the X and Y chromosomes, were microdissected. The DNA was PCR amplified, non-radioactively labelled and used as probes on equine metaphase chromosomes to confirm their origin. Once tested, the paints were used as probes on donkey metaphase chromosomes to detect homologous chromosomal segments between the two species. The results not only detected conservation of whole chromosome and/or arm ...
Multiple DNA markers differentiate Sarcocystis neurona and Sarcocystis falcatula. Studies designed to investigate the causative agent of equine protozoal myeloencephalitis and its life cycle have been hampered by the marked similarity of Sarcocystis neurona to other Sarcocystis spp. present in the same definitive host. Random-amplified polymorphic DNA techniques were used to amplify DNA from isolates of S. neurona and Sarcocystis falcatula. DNA sequence analysis of polymerase chain reaction (PCR) products was then used to design PCR primers to amplify specific Sarcocystis spp. DNA products. The ribosomal RNA internal transcribed spacer was also amplified and compared betwee...
Endotoxin induction of nitric oxide synthase and cyclooxygenase-2 in equine alveolar macrophages. To determine the amount of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) enzymes induced in vitro in equine alveolar macrophages in response to lipopolysaccharide (LPS). Sample Population-Alveolar macrophages obtained from 12 horses. Methods: Alveolar macrophages were collected by bronchoalveolar lavage from 12 horses and incubated for 6 hours with LPS (0.001 to 10 microg/ml) or vehicle. Total RNA was extracted and purified. After first-strand cDNA synthesis, mRNA induction was measured, using a polymerase chain reaction (PCR) technique for COX-2, iNOS, and glyceraldehyde...
A sensitive polymerase chain reaction based assay for the detection of Setaria digitata: the causative organism of cerebrospinal nematodiasis in goats, sheep and horses. A sensitive PCR assay for the detection of Setaria digitata has been developed. Two oligonucleotide primers (17 nt) were designed from a previously cloned and characterized tandemly arranged repetitive sequence of Setaria digitata. Using these primers, it was possible to amplify small quantities (100 fg) of S. digitata genomic DNA. A simple procedure, using proteinase K and non-ionic detergent NP 40, was followed to process the host blood samples and mosquitoes harbouring L3 larvae. The sensitivity of the polymerase chain reaction based assay surpasses the microscopic detection and the previou...
Comparative mapping of 18 equine type I genes assigned by somatic cell hybrid analysis. Polymerase chain reaction primers designed from horse cDNA sequences and from consensus sequences highly conserved in mammalian species were used to amplify markers for synteny mapping 18 equine type I genes. These markers were used to screen a horse-mouse somatic cell hybrid panel (UCDavis SCH). Fourteen primer sets amplified horse-specific fragments, while restriction enzyme digests of PCR products were used to distinguish the fragments amplified from horse and mouse with four primer sets. Synteny assignments were made based on correlation values between each marker tested and other markers ...
Quantitative measurement of equine cytokine mRNA expression by polymerase chain reaction using target-specific standard curves. Quantification of cytokine mRNA using reverse transcription coupled with the polymerase chain reaction (RT-PCR) has become a corner stone of the study of cytokine regulation. Quantitative competitive RT-PCR (QCRT-PCR) is commonly accepted as a reliable method for quantifying differences in mRNA levels but is both labor- and reagent-intensive. A noncompetitive polymerase chain reaction method that utilizes cytokine-specific, plasmid-derived, standard curves was developed for the quantification of equine cytokine mRNA. The assay can be performed on minute samples of cellular material, utilizes s...
Quantitation of equine cytokine mRNA expression by reverse transcription-competitive polymerase chain reaction. A reverse transcription-competitive polymerase chain reaction (RT-cPCR) method was developed to quantitate equine interleukin (IL)-1alpha, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 p35, IL-12 p40, interferon-gamma (INF-gamma), tumor necrosis factor-alpha (TNF-alpha), and beta-actin mRNA expression. Using primers based on equine-specific sequences, these cytokines could be detected in concanavalin A-stimulated peripheral blood mononuclear cells. The specificity of the amplified product was confirmed by sequencing. For each cytokine, the assay was made quantitative by generating competitor ...
Prevalence of beta2-toxigenic Clostridium perfringens in horses with intestinal disorders. The incidence of a new, yet unassigned toxin type of Clostridium perfringens containing the genes for the alpha-toxin and the recently described beta2-toxin in horses with intestinal disorders is reported. The study included 18 horses suffering from typical typhlocolitis, 7 horses with atypical typhlocolitis, 16 horses with other intestinal disorders, and 58 horses without intestinal disease. In total, 20 samples of ingesta of the small and large intestines, five biopsy specimens of the intestinal wall, and 74 fecal samples were analyzed bacteriologically. C. perfringens isolates were typed fo...
An equine herpesvirus 1 mutant with a lacZ insertion between open reading frames 62 and 63 is replication competent and causes disease in the murine respiratory model. An equine herpesvirus 1 (EHV-1) mutant was constructed by inserting a lacZ expression cassette into the intergenic region upstream of gene 62 (glycoprotein L; gL) and downstream of gene 63 (a homologue of the herpes simplex virus transcriptional activator ICP0). The recombinant lacZ62/63-EHV-1 had similar growth kinetics in cell culture to those of the parental wild type (wt) virus, with indistinguishable cytopathic effects and plaque morphology. Reverse transcriptase PCR confirmed that the lacZ insertion did not interfere with transcription of gL and immunoblot analysis indicated there was no...
Experimental infection of four horses with Ehrlichia phagocytophila. Four clinically healthy horses which were negative for antibodies to Ehrlichia phagocytophila, the agent of bovine ehrlichiosis, were infected experimentally with E phagocytophila-containing bovine leucocytes, administered intravenously. The horses were examined daily for four weeks, and blood samples were collected daily for cytological, haematological and biochemical examination and for a nested polymerase chain reaction (PCR). An indirect immunofluorescence test was used to determine when the horses seroconverted and the duration of positive titres. There were no abnormal clinical, haematol...
Use of reverse transcriptase-polymerase chain reaction (RT-PCR) and dot-blot hybridisation for the detection and identification of African horse sickness virus nucleic acids. A coupled reverse transcriptase-polymerase chain reaction assay (RT-PCR) for the detection of African horse sickness virus (AHSV) dsRNA, has been developed using genome segment 7 as the target template for primers. RNA from isolates of all nine AHSV serotypes were readily detected. The potential inhibitory effects of either ethylene diamine tetra acetic acid (EDTA) or heparin on the RT-PCR were eliminated by washing blood samples before lysis of the red blood cells and storage. There was a close agreement in the sensitivity and the specificity of the RT-PCR and an indirect sandwich ELISA. Conf...
Clinical, serologic, and histopathologic characterization of experimental Borna disease in ponies. Borna disease was originally described as an equine neurologic syndrome over 200 years ago, although the infectious etiology of the disorder was unproven until the early 20th century. Borna disease virus (BDV) was finally isolated from horses dying of the disorder, and that virus has been used to experimentally reproduce Borna disease in several species of laboratory animals. However, BDV has never been inoculated back into horses to experimentally and etiologically confirm the classic clinical, pathologic, and serologic characteristics of the disease in that species. Three ponies were intrace...
Experimental infection of the human granulocytic ehrlichiosis agent in horses. Human blood collected from two patients from Westchester County, New York with human granulocytic ehrlichia (HGE) infection was inoculated into two ponies. Inoculated ponies developed clinical signs similar to a previous report (Madigan et al., 1995). Histopathological changes involved follicular hyperplasia of lymphoid tissues. HGE DNA was detected by PCR in muscle, fascia, peritoneum, and adrenal gland after the ponies produced a high level of antibodies to HGE. We suggest that HGE may reside in poorly vascularized connective tissues, where the antibodies may have some difficulties to penetr...
Virological and molecular biological investigations into equine herpes virus type 2 (EHV-2) experimental infections. Two 18-month-old naturally reared ponies were used to investigate the pathogenicity of EHV-2. After dexamethasone treatment, pony 1 was inoculated intranasally with EHV-2 strain T16, which has been isolated from a foal with keratoconjunctivitis superficialis and pony 2 was similarly inoculated with strain LK4 which was originally isolated from a horse with upper respiratory tract disease. Following virus inoculation, pyrexia was not detected in either pony but both developed conjunctivitis, lymphadenopathy, and coughing. EHV-2 was detected in nasal mucus samples up to day 12 post infection (p....
Equine infectious anemia virus is found in tissue macrophages during subclinical infection. The equine infectious anemia virus (EIAV) often results in lifelong subclinical infection following early episodes of clinical disease. To identify the cellular reservoirs of EIAV during subclinical infection, horses were infected with EIAV and allowed to develop subclinical infections. Horses with acute disease served as a basis for comparison. The tissue distribution, replication status, location of infected cells, and viral load were characterized by PCR for proviral DNA and reverse transcriptase PCR for viral RNA, in situ hybridization, and in situ PCR. Proviral DNA was widespread in tissu...
