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Topic:DNA
DNA in horses refers to the genetic material that carries the hereditary information necessary for the growth, development, functioning, and reproduction of equine species. It consists of sequences of nucleotides that encode the genetic instructions used in the development and functioning of horses. DNA analysis in horses can provide insights into genetic diversity, lineage, and breed characteristics. It is also utilized in identifying genetic disorders, understanding hereditary traits, and assisting in selective breeding programs. This page compiles peer-reviewed research studies and scholarly articles that explore the structure, function, and applications of DNA analysis in equine genetics and breeding.
Comparison of concentrations of Rhodococcus equi and virulent R. equi in air of stables and paddocks on horse breeding farms in a temperate climate. Rhodococcoccus equi is a significant cause of bronchopneumonia in foals worldwide. Infection of the lungs is believed to result from inhalation of virulent R. equi in dust from contaminated environments. A measure of infectious risk in an environment is the level of airborne contamination. Objective: To assess and compare the level of airborne virulent R. equi in paddocks and stables. Methods: Air samples were collected sequentially over the 2003 foaling season from the paddocks and stables on 3 Irish horse breeding farms affected by R. equi pneumonia. Colony blotting and DNA hybridisation tec...
Quantification by real-time PCR of the magnitude and duration of leucocyte-associated viraemia in horses infected with neuropathogenic vs. non-neuropathogenic strains of EHV-1. 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...
TNF-alpha SNP haplotype frequencies in equidae. Tumour necrosis factor alpha (TNF-alpha) is a pro-inflammatory cytokine that plays a crucial role in the regulation of inflammatory and immune responses. In all vertebrate species the genes encoding TNF-alpha are located within the major histocompatability complex. In the horse TNF-alpha has been ascribed a role in a variety of important disease processes. Previously two single nucleotide polymorphisms (SNPs) have been reported within the 5' un-translated region of the equine TNF-alpha gene. We have examined the equine TNF-alpha promoter region further for additional SNPs by analysing DNA from...
Extended phylogeny of equine arteritis virus: division into new subgroups. To determine a conclusive phylogeny, equine arteritis viruses from Italy, Austria, Hungary, Sweden, South Africa and other parts of the world were analysed by reverse-transcription polymerase chain reaction amplification and direct sequencing. The nucleotide sequences corresponding to the variable part of the large glycoprotein GP5, specified by open reading frame 5, were compared and added to a previously published phylogenetic tree in which a clear division between 'European' and 'American' type viruses had been established. Adding the sequences determined in this study and new sequences ret...
Identification of pathogenic Leptospira strains in tissues of a premature foal by use of polymerase chain reaction analysis. Studies were carried out to determine the cause of death in a prematurely born Thoroughbred foal that died 24 hours after birth. Necropsy revealed gross lesions suggestive of septicemia. A commercial Leptospira polymerase chain reaction (PCR) assay designed to specifically amplify the hemolysis-associated protein 1 (hap1) gene present only in pathogenic Leptospira strains detected the presence of Leptospira DNA in various tissues of the foal. Histologic examination of lung, liver, kidney, and myocardium revealed numerous spirochetes in Warthin-Starry-stained tissue sections. Results of PCR ana...
Internal restriction sites: quality assurance aids in genotyping. Improvements to restriction fragment length polymorphism (RFLP)-based genotyping assays currently used for detection of mutations responsible for bovine ferrochelatase and myophosphorylase deficiencies, and equine hyperkalemic periodic paralysis (HYPP) are described. Reports of sporadic inhibition of restriction enzyme activity suggest a critical factor in RFLP-based genotyping assays should be assurance that restriction enzymes perform to specification with every sample. The RFLP genotyping assays that use either a mismatched recognition sequence in one or both of the oligonucleotides, or inc...
A human-horse comparative map based on equine BAC end sequences. In an effort to increase the density of sequence-based markers for the horse genome we generated 9473 BAC end sequences (BESs) from the CHORI-241 BAC library with an average read length of 677 bp. BLASTN searches with the BESs revealed 4036 meaningful hits (E <or= 10(-5)) in the human genome that provide useful markers for the human-horse comparative map. The 4036 BLASTN hits allowed the anchoring of 3079 BAC clones to the human genome, on average one corresponding equine BAC clone per megabase of human DNA. We used the BLASTN anchored BESs for an in silico prediction of the gene content an...
Malassezia species isolated from the intermammary and preputial fossa areas of horses. Malassezia-type yeasts previously have been observed on cytologic examination of the intermammary region of mares that presented with tail-head pruritus; topical antiyeast treatment resolved the pruritus. Further, Malassezia dermatitis has been observed in horses in intertriginous areas such as the udder and prepuce; the species of yeast was not confirmed. It is not known whether healthy mares or male horses can be carriers of this yeast in these body areas. Objective: Malassezia spp. are present in the intermammary region in healthy mares and the preputial fossa in healthy geldings. Methods: ...
Sequence analysis of a 212 kb defensin gene cluster on ECA 27q17. Defensins are a family of evolutionary ancient antimicrobial peptides consisting of three sub-families: alpha-, beta- and theta-defensins. This investigation was focused on the genomic characterization of equine beta-defensins and the investigation of the potential clustering of beta-defensin genes in the equine genome. Six genomic BAC clones were isolated from the CHORI-241 library and one of these was mapped by FISH to ECA 27q17. This location was confirmed by RH-mapping. The contiguous 212 kb sequence of this clone was determined. Sequence analysis revealed the identification of ten pseudog...
Construction of a medium-density horse gene map. A medium-density map of the horse genome (Equus caballus) was constructed using genes evenly distributed over the human genome. Three hundred and twenty-three exonic primer pairs were used to screen the INRA and the CHORI-241 equine BAC libraries by polymerase chain reaction and by filter hybridization respectively. Two hundred and thirty-seven BACs containing equine gene orthologues, confirmed by sequencing, were isolated. The BACs were localized to horse chromosomes by fluorescent in situ hybridization (FISH). Overall, 165 genes were assigned to the equine genomic map by radiation hybrid (RH...
Use of DNA and recombinant canarypox viral (ALVAC) vectors for equine herpes virus vaccination. In this study, experimental canarypox virus (ALVAC) and plasmid DNA recombinant vaccines expressing the gB, gC and gD glycoproteins of EHV-1 were assessed for their ability to protect conventional ponies against a respiratory challenge with EHV-1. In addition, potential means of enhancing serological responses in horses to ALVAC and DNA vaccination were explored. These included co-administration of the antigen with conventional adjuvants, complexation with DMRIE-DOPE and co-expression of the antigen along with equine GM-CSF. Groups of EHV primed ponies were vaccinated twice intra-muscularly wi...
Analysis of equid herpesvirus 1 strain variation reveals a point mutation of the DNA polymerase strongly associated with neuropathogenic versus nonneuropathogenic disease outbreaks. Equid herpesvirus 1 (EHV-1) can cause a wide spectrum of diseases ranging from inapparent respiratory infection to the induction of abortion and, in extreme cases, neurological disease resulting in paralysis and ultimately death. It has been suggested that distinct strains of EHV-1 that differ in pathogenic capacity circulate in the field. In order to investigate this hypothesis, it was necessary to identify genetic markers that allow subgroups of related strains to be identified. We have determined all of the genetic differences between a neuropathogenic strain (Ab4) and a nonneuropathogenic ...
[Characterization of Rickettsia spp. circulating in a silent peri-urban focus for Brazilian spotted fever in Caratinga, Minas Gerais, Brazil]. The present study was intended to characterize Rickettsia spp. circulating in arthropod vectors in Caratinga, Minas Gerais, Brazil, by PCR and to investigate the presence of antibodies against the spotted fever Rickettsiae group (SFRG) in dogs and horses. 2,610 arthropods were collected and taxonomically identified. DNA samples obtained from these vectors were submitted to PCR and cycle-sequenced. Ctenocephalides and Amblyomma cajennense showed sequences presenting 100.0% homology with R. felis. A sequence obtained from Rhipicephalus sanguineus showed 99.0% homology with R. felis, and a sequen...
Antibody and cellular immune responses following DNA vaccination and EHV-1 infection of ponies. Equine herpesvirus-1 (EHV-1) is the cause of serious disease with high economic impact on the horse industry, as outbreaks of EHV-1 disease occur every year despite the frequent use of vaccines. Cytotoxic T-lymphocytes (CTLs) are important for protection from primary and reactivating latent EHV-1 infection. DNA vaccination is a powerful technique for stimulating CTLs, and the aim of this study was to assess antibody and cellular immune responses and protection resulting from DNA vaccination of ponies with combinations of EHV-1 genes. Fifteen ponies were divided into three groups of five ponies...
Mitochondrial DNA: an important female contribution to thoroughbred racehorse performance. The mitochondrial DNA (mtDNA) molecule, carrying genes encoding for respiratory chain enzymes, is a primary candidate for demonstrating associations between genotype and athletic performance in mammalian species. In humans, variation at seven protein encoding mitochondrial loci has been implicated in influencing fitness and performance characteristics. Although thoroughbred horses are selected for racing ability, there have not been any previous reported associations between genotypes and racecourse performance. The multi-factorial nature of the inheritance of racing ability is an obvious comp...
A 1.3-Mb interval map of equine homologs of HSA2. A comparative approach that utilizes information from more densely mapped or sequenced genomes is a proven and efficient means to increase our knowledge of the structure of the horse genome. Human chromosome 2 (HSA2), the second largest human chromosome, comprising 243 Mb, and containing 1246 known genes, corresponds to all or parts of three equine chromosomes. This report describes the assignment of 140 new markers (78 genes and 62 microsatellites) to the equine radiation hybrid (RH) map, and the anchoring of 24 of these markers to horse chromosomes by FISH. The updated equine RH maps for ECA...
Cloning and expression of 51-kDa antigenic protein of Neorickettsia risticii NR-JA1. Neorickettsia (Ehrlichia) risticii is a causative agent of acute diarrheal syndrome in horses, commonly known as Potomac horse fever. Korean isolate of N. risticii NR-JA1 was cultivated in mouse macrophage cell line P388D1. A complete ORF of p51 antigenic protein gene was amplified and cloned into pQE32 and pcDNA3.1 vectors and the resultant clones were named as pQE32/Nr-51 and pcDNA3.1/Nr-51, respectively. Recombinant p51 (rp51) protein antigen was expressed in E. coli (pQE32/Nr-51) and cos-7 cell line (pcDNA3.1/Nr-51). The rp51 protein showed immunoreactivity with anti- mouse p51 antibodies....
Iberian origins of New World horse breeds. Fossil records, archaeological proofs, and historical documents report that horses persisted continuously in the Iberian Peninsula since the Pleistocene and were taken to the American continent (New World) in the 15th century. To investigate the variation within the mitochondrial DNA (mtDNA) control region of Iberian and New World horse breeds, to analyze their relationships, and to test the historical origin of New World horses, a total of 153 samples, representing 30 Iberian and New World breeds, were analyzed by sequencing mtDNA control region fragments. Fifty-four haplotypes were found and...
Equine herpesvirus-4 kinetics in peripheral blood leukocytes and nasopharyngeal secretions in foals using quantitative real-time TaqMan PCR. Based on the hypothesis that the viral load of cells infected with EHV-4 will likely change during the course of disease, TaqMan PCR was used to investigate and characterize the kinetics of EHV-4 viral DNA load (glycoprotein B gene) and transcriptional activity (glycoprotein B and latency-associated transcripts) in peripheral blood leukocytes (PBLs) and nasopharyngeal secretions (NSs) collected from 11 foals during a field outbreak of respiratory disease. The EHV-4 DNA load in PBLs was low and of short duration after onset of clinical signs. In contrast, the EHV-4 load in NSs remained high for...
Genotyping of Toll-like receptor 4, myeloid differentiation factor 2 and CD-14 in the horse: an investigation into the influence of genetic polymorphisms on the LPS induced TNF-alpha response in equine whole blood. The inter- and intra-species differences in the response to lipopolysaccharides (LPS) are well recognised in mammalian species. It has been hypothesized that these differences can be attributed to genetic polymorphisms in the components involved in LPS signal transduction. These components include the cluster of differentiation factor 14 (CD-14), a membrane bound protein on the surface of mononuclear cells that recognises LPS and a receptor complex consisting of Toll-like receptor-4 (TLR-4) and myeloid differentiation factor-2 (MD-2). Sequencing of these three proteins in humans and mice revea...
Detection of the reemerging agent Burkholderia mallei in a recent outbreak of glanders in the United Arab Emirates by a newly developed fliP-based polymerase chain reaction assay. A polymerase chain reaction (PCR) assay targeting the flagellin P (fliP)-I S407A genomic region of Burkholderia mallei was developed for the specific detection of this organism in pure cultures and clinical samples from a recent outbreak of equine glanders. Primers deduced from the known fliP-IS407A sequence of B. mallei American Type Culture Collection (ATCC) 23344(T) allowed the specific amplification of a 989-bp fragment from each of the 20 B. mallei strains investigated, whereas other closely related organisms tested negative. The detection limit of the assay was 10 fg for purified DNA of ...
Effect of microcurrent electrical tissue stimulation on equine tenocytes in culture. To determine effects of microcurrent electrical tissue stimulation (METS) on equine tenocytes cultured from the superficial digital flexor tendon (SDFT). Methods: SDFTs were collected from 20 horses at slaughter. Methods: Tenocytes were isolated following outgrowth from explants and grown in 48-well plates. Four methods of delivering current to the tenocytes with a METS device were tested. Once the optimal method was selected, current consisting of 0 (negative control), 0.05, 0.1, 0.5, 1.0, or 1.5 mA was applied to cells (8 wells/current intensity) once daily for 8 minutes. Cells were treated ...
Sequence variation of the SeM gene of Streptococcus equi allows discrimination of the source of strangles outbreaks. Improved understanding of the epidemiology of Streptococcus equi transmission requires sensitive and portable subtyping methods that can rationally discriminate between strains. S. equi is highly homogeneous and cannot be distinguished by multilocus enzyme electrophoretic or multilocus sequence-typing methods that utilize housekeeping genes. However, on sequence analysis of the N-terminal region of the SeM genes of 60 S. equi isolates from 27 strangles outbreaks, we identified 21 DNA codon changes. These resulted in the nonsynonymous substitution of 18 amino acids and allowed the assignment of...
Molecular diagnosis and equine parasitology. The future implementation of improved and sustainable control strategies for the major equine parasites will be dependent on a greater insight into their basic biology, pathogenicity and epidemiology together with an enhanced ability for accurate diagnosis. This paper will provide a review of the current molecular methods under development for the detection of equine parasites and their application to current scientific questions. In particular, the strongyles are recognised as important pathogens of horses and recent advances made in the study of this parasitic group at the single species lev...
Nuclear status of immature and mature stallion spermatozoa. 'The highly packed chromatin of mature spermatozoa results from replacement of somatic-like histones by highly basic arginine- and cysteine-rich protamines during spermatogenesis, with additional conformational changes in chromatin structure during epididymal transit. The objective of the present study was to compare the nuclear characteristics of immature and mature epididymal stallion spermatozoa, using a variety of experimental approaches. Resistance to in vitro decondensation of chromatin, following exposure to SDS-DTT and alkaline thioglycolate, increased significantly in mature spermatoz...
Molecular diagnosis of anthelmintic resistance. Conventional and real time polymerase chain reaction-based tests have been developed for the diagnosis of anthelmintic resistance (AR) in populations of several small and large ruminant as well as horse gastro-intestinal nematode species. To date, molecular markers that correlate well with AR are available only for the detection of benzimidazole resistance. Recently, however, a single nucleotide polymorphism was found in vitro to be of functional relevance for reduced drug efficacy to macrocylic lactones. The focus of the present review, therefore, is the molecular mechanism of action of these...
Evaluation of a nested PCR test and bacterial culture of swabs from the nasal passages and from abscesses in relation to diagnosis of Streptococcus equi infection (strangles). Streptococcus equi is the cause of strangles in horses. To improve diagnostic sensitivity, development and evaluation of DNA-based methods are necessary. Objective: To evaluate diagnostic methods and observe the pattern of bacterial shedding during natural outbreaks. Methods: Two herds with natural outbreaks of strangles were visited over a period of 15 weeks and 323 samples originating from 35 horses investigated. The diagnostic use of a nested PCR test was evaluated using a collection of 165 isolates of Lancefield group C streptococci (species specificity) and swabs from nasal passages or fr...
Evolutionary movement of centromeres in horse, donkey, and zebra. Centromere repositioning (CR) is a recently discovered biological phenomenon consisting of the emergence of a new centromere along a chromosome and the inactivation of the old one. After a CR, the primary constriction and the centromeric function are localized in a new position while the order of physical markers on the chromosome remains unchanged. These events profoundly affect chromosomal architecture. Since horses, asses, and zebras, whose evolutionary divergence is relatively recent, show remarkable morphological similarity and capacity to interbreed despite their chromosomes differing co...
Characterization of the cDNA Encoding alphaIIb and beta3 in normal horses and two horses with Glanzmann thrombasthenia. Glanzmann thrombasthenia (GT) is an inherited, intrinsic platelet defect characterized by a quantitative or qualitative change in the platelet glycoprotein complex IIb-IIIa (integrin alpha(IIb)beta3). The subunits are encoded by separate genes and both subunits must be expressed for a stable complex to form on the platelet surface; therefore, a defect in either gene can result in GT.
