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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.
Proviral genomic sequence analysis of Chinese donkey leukocyte attenuated equine infectious anemia virus vaccine and its parental virus strain Liaoning. Proviral DNA was extracted from donkey leukocyte infected with Chinese donkey leukocyte attenuated equine infectious anemia virus (DLA-EIAV), and peripheral blood lymphocytes (PBL) from a horse infected with the virulent EIAV strain Liaoning (EIAV L). The entire proviral DNA from both viruses was cloned and sequenced. The lengths of complete genomic sequences of DLA-EIAV and EIAV L provirus were 8266 bp and 8235 bp, respectively. Sequence comparison indicated that DLA-EIAV shares 97.0% and 97.5% in sequence homology with EIAV L and donkey-adapted EIAV (DA-EIAV), respectively. Lots of variation...
Isolation and analysis of the genetic diversity of repertoires of VSG expression site containing telomeres from Trypanosoma brucei gambiense, T. b. brucei and T. equiperdum. African trypanosomes (including Trypanosoma brucei) are unicellular parasites which multiply in the mammalian bloodstream. T. brucei has about twenty telomeric bloodstream form Variant Surface Glycoprotein (VSG) expression sites (BESs), of which one is expressed at a time in a mutually exclusive fashion. BESs are polycistronic transcription units, containing a variety of families of expression site associated genes (ESAGs) in addition to the telomeric VSG. These polymorphic ESAG families are thought to play a role in parasite-host adaptation, and it has been proposed that ESAG diversity might ...
Detection of equine herpesvirus-1 in nasal swabs of horses by quantitative real-time PCR. Early identification of inhalation-transmitted equine herpesvirus type 1 (EHV-1) infections has been facilitated by the availability of a number of real-time quantitative PCR (qPCR) tests. A direct comparison between nasal swab qPCR and traditional virus isolation (VI) requires a method for normalizing the qPCR samples and controlling for PCR inhibitors present in some clinical samples. Objective: To quantify EHV-1 shedding in viral swabs using an internal control and to compare fast qPCR to VI for the detection of EHV-1 in nasal swabs from horses. Methods: Fifteen horses experimentally infect...
Glycogen synthase 1 (GYS1) mutation in diverse breeds with polysaccharide storage myopathy. A missense mutation in the GYS1 gene was recently described in horses with polysaccharide storage myopathy (PSSM). Objective: The first objective was to determine the prevalence of the GYS1 mutation in horses with PSSM from diverse breeds. The second objective was to determine if the prevalence of the GYS1 mutation differed between horses diagnosed with PSSM based on grade 1 (typically amylase-sensitive) or grade 2 (typically amylase-resistant) polysaccharide. Methods: Eight hundred and thirty-one PSSM horses from 36 breeds. Methods: Horses with PSSM diagnosed by histopathology of skeletal mus...
Effect of dexamethasone supplementation on chondrogenesis of equine mesenchymal stem cells. To determine whether expansion of equine mesenchymal stem cells (MSCs) by use of fibroblast growth factor-2 (FGF-2) prior to supplementation with dexamethasone during the chondrogenic pellet culture phase would increase chondrocytic matrix markers without stimulating a hypertrophic chondrocytic phenotype. Methods: MSCs obtained from 5 young horses. Methods: First-passage equine monolayer MSCs were supplemented with medium containing FGF-2 (0 or 100 ng/mL). Confluent MSCs were transferred to pellet cultures and maintained in chondrogenic medium containing 0 or 10(7)M dexamethasone. Pellets were...
Genetic analysis of the Hispano-Breton heavy horse. Hispano-Breton (HB) is a horse breed with a recent mixed ancestry. It was developed in the 1930s by crossing local mares with Breton draught horses imported from France. Nowadays it is considered to be in a vulnerable situation due to census decline. To genetically characterize the breed and to set up the basis for a conservation programme, we have employed two types of molecular markers: a 347-bp D-loop mitochondrial DNA (mtDNA) fragment and 13 microsatellite loci. A representative sample of 53 HB individuals was analysed together with a sample of 40 Pura Raza Española horses for comparison....
Equine synovial fluid analysis. The most important application for synovial fluid (SF) analysis in the horse is in the diagnosis of synovial sepsis. Misdiagnosis of synovial sepsis is costly, and SF analysis makes correct diagnosis more likely, although far from certain. The precision of diagnosis may be increased with polymerase chain reaction analysis for detection of bacterial DNA in SF and with assays for various enzymes and cytokines. These tests are currently not widely available, however, and routine SF analysis remains of prime importance in diagnosis.
Complementary DNA cloning, functional expression and characterization of a novel cytochrome P450, CYP2D50, from equine liver. Members of the CYP2D family constitute only about 2-4% of total hepatic CYP450s, however, they are responsible for the metabolism of 20-25% of commonly prescribed therapeutic compounds. CYP2D enzymes have been identified in a number of different species. However, vast differences in the metabolic activity of these enzymes have been well documented. In the horse, the presence of a member of the CYP2D family has been suggested from studies with equine liver microsomes, however its presence has not been definitively proven. In this study a cDNA encoding a novel CYP2D enzyme (CYP2D50) was cloned f...
A cis-acting regulatory mutation causes premature hair graying and susceptibility to melanoma in the horse. In horses, graying with age is an autosomal dominant trait associated with a high incidence of melanoma and vitiligo-like depigmentation. Here we show that the Gray phenotype is caused by a 4.6-kb duplication in intron 6 of STX17 (syntaxin-17) that constitutes a cis-acting regulatory mutation. Both STX17 and the neighboring NR4A3 gene are overexpressed in melanomas from Gray horses. Gray horses carrying a loss-of-function mutation in ASIP (agouti signaling protein) had a higher incidence of melanoma, implying that increased melanocortin-1 receptor signaling promotes melanoma development in Gra...
Substitution of human for horse urine disproves an accusation of doping*. In order to detect switching and/or manipulation of samples, the owner of a stallion asked our lab to perform a DNA test on a positive doping urine sample. The objective was to compare the urine DNA profile versus blood and hair DNA profiles from the same stallion. At first, 10 microsatellite markers were investigated to determine the horse identity. No results were obtained when horse specific markers were typed in the urine sample. In order to confirm the species origin of this sample we analyzed the mitochondrial cytochrome b gene. This analysis from blood and hair samples produced reproduc...
Gonadotropin-dependent regulation of the prostaglandin E2 receptor in equine preovulatory follicles during the ovulatory process in mares. The objectives of the study were to clone the primary structure of the prostaglandin E2 receptor subtype 2 (PTGER2) cDNA and to characterize its regulation in equine follicles during gonadotropin-induced ovulation. Results from DNA isolation indicated that the equine PTGER2 cDNA encodes a predicted 353-amino acid protein, which is highly similar (76-85%) to known mammalian homologues. The regulation of PTGER2 was studied by semi-quantitative RT-PCR/Southern blot using preparations of theca interna and mural granulosa cells isolated from equine follicles 0-39 hr post-treatment with human chorio...
The horse pseudoautosomal region (PAR): characterization and comparison with the human, chimp and mouse PARs. The pseudoautosomal region (PAR) is a genomic segment on mammalian sex chromosomes where sequence homology mimics that seen between autosomal homologues. The region is essential for pairing and proper segregation of sex chromosomes during male meiosis. As yet, only human/chimp and mouse PARs have been characterized. The two groups of species differ dramatically in gene content and size of the PAR and therefore do not provide clues about the likely evolution and constitution of PAR among mammals. Here we characterize the equine PAR by i) isolating and arranging 71 BACs containing 129 markers (1...
Helicobacter equorum is highly prevalent in foals. Faecal samples of sixty-six 3-day- to 6-month-old foals were screened for Helicobacter equorum DNA by means of a PCR amplifying a 1074 bp fragment of the 23S rRNA gene with primers specific for this enterohepatic Helicobacter species. H. equorum DNA was demonstrated in faeces from 28.6% of the less than 1-month-old foals, while 67.8% of foals from 1 to 6 months of age tested positive. In a previous study, H. equorum was demonstrated in faeces of 0.8-7.9% of adult horses. These results indicate that the prevalence of H. equorum in horses differs with the age of the investigated horse population...
Haplotypic variability within the mitochondrial gene encoding for the cytochrome c oxidase 1 (cox1) of Cylicocyclus nassatus (Nematoda, Strongylida): evidence for an affiliation between parasitic populations and domestic and wild equid hosts. This study investigated the genetic variability within Cylicocyclus nassatus (Nematoda, Strongylida, Cyathostominae) collected from different domestic and wild hosts (i.e. horse, donkey, Przewalskii horse, tarpan and Turkmen kulan) and localities in Europe and/or USA. The ribosomal Internal Transcribed Spacer 2 (ITS2) and the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were PCR-amplified and sequences characterized from seventy individual parasitic specimens. While ITS2 displayed 0-0.6% variation rate among all individual adult specimens of C. nassatus examined, 22 different seque...
Evaluation of a multiplex PCR for detection of serotypes K1, K2 and K5 in Klebsiella sp. and comparison of isolates within these serotypes. A multiplex PCR using targets within the serotype-specific region of the capsular polysaccharide synthesis gene cluster of serotypes K1, K2 and K5 was evaluated using the 77 reference serotype strains of Klebsiella, and a panel of clinical isolates subjected previously to conventional serotyping. The PCR was highly specific for these serotypes, which are those most associated with virulence in humans and horses. PCR confirmed that isolates of the K5 serotype had cross-reacted with antiserum for other serotypes, particularly for K7. K5 isolates received by our laboratory were almost exclusively...
Estrogenic activity of the equine estrogen metabolite, 4-methoxyequilenin. Oxidative metabolism of estrogens has been associated with genotoxicity. O-methylation of catechol estrogens is considered as a protective mechanism. 4-Methoxyequilenin (4-MeOEN) is the O-methylated product of 4-hydroxyequilenin (4-OHEN). 4-OHEN, the major catechol metabolite of the equine estrogens present in the most widely prescribed hormone replacement therapeutics, causes DNA damage via quinone formation. In this study, estrogen receptor (ERa) binding of 4-MeOEN was compared with estradiol (E2) and equilenin derivatives including 4-BrEN using computer modeling, estrogen response element (...
[Hereditary diseases in the horse: I. Monogenetic diseases]. Overall, monogenetic hereditary diseases are less important for the breeding industry than polygenetic diseases because they are relatively rare. For the individual animal, however, these diseases have often a dramatic outcome and many of these diseases presently known are lethal. For several of them the exact pathogenesis is known and DNA-tests are available to confirm the exact diagnosis.
Peripheral blood mononuclear cells represent a reservoir of bovine papillomavirus DNA in sarcoid-affected equines. Bovine papillomaviruses of types 1 and 2 (BPV-1 and -2) chiefly contribute to equine sarcoid pathogenesis. However, the mode of virus transmission and the presence of latent infections are largely unknown. This study established a PCR protocol allowing detection of <or=10 copies of the BPV-1/-2 genes E5 and L1. Subsequent screening of peripheral blood mononuclear cell (PBMC) DNA derived from horses with and without BPV-1/2-induced skin lesions demonstrated the exclusive presence of E5, but not L1, in PBMCs of BPV-1/2-infected equines. To validate this result, a blind PCR was performed from ...
A BAC contig map over the proximal approximately 3.3 Mb region of horse chromosome 21. A total of 207 BAC clones containing 155 loci were isolated and arranged into a map of linearly ordered overlapping clones over the proximal part of horse chromosome 21 (ECA21), which corresponds to the proximal half of the short arm of human chromosome 19 (HSA19p) and part of HSA5. The clones form two contigs - each corresponding to the respective human chromosomes - that are estimated to be separated by a gap of approximately 200 kb. Of the 155 markers present in the two contigs, 141 (33 genes and 108 STS) were generated and mapped in this study. The BACs provide a 4-5x coverage of the regio...
A case of an intersex horse with 63,X/64,XX/65,XX,del(Y)(q?) karyotype. Cytogenetic and molecular genetic studies of an intersex horse have been carried out. The investigated animal had overall male body conformation; however, its external genitalia consisted of incompletely developed vulva and penis. The X and Y chromosome painting probes detected three cell lines in the examined horse: 63,X, 64,XX and 65,XX with a fragment of a Y chromosome (del Y). The DNA analysis with the PCR and PCR/RFLP methods showed absence of SRY,AMELY and ZFY genes as well as of six Y microsatellite markers (YM2, YP9, YJ10, YE1, YH12, and YA16). These results suggest that the Y chromoso...
Single-round infectious particles enhance immunogenicity of a DNA vaccine against West Nile virus. DNA vaccines encoding replication-defective viruses are safer than inactivated or live attenuated viruses but may fail to stimulate an immune response sufficient for effective vaccination. We augment the protective capacity of a capsid-deleted flavivirus DNA vaccine by co-expressing the capsid protein from a separate promoter. In transfected cells, the capsid-deleted RNA transcript is replicated and translated to produce secreted virus-like particles lacking the nucleocapsid. This RNA is also packaged with the help of co-expressed capsid protein to form secreted single-round infectious particl...
Apoptotic-like changes in equine spermatozoa separated by density-gradient centrifugation or after cryopreservation. The objective was to evaluate apoptotic markers in ejaculated equine spermatozoa after separation by density-gradient centrifugation and after cryopreservation. Subpopulations of percoll-separated equine spermatozoa differed (P<0.05) in the percentage of live, caspase-activated spermatozoa (2.9+/-0.7% vs 14.2+/-6.4%; mean+/-S.E.M.), low mitochondrial membrane potential (MMP; 6.8+/-1.1 vs 23.8+/-3.7), altered plasma membrane permeability (1.3+/-0.2 vs 3.0+/-0.5), DNA fragmentation (2.0+/-1.3 vs 14.3+/-3.6), total motility (81.8+/-3.3 vs 35.1+/-5.4), and progressive motility (66.3+/-4.3 vs 24...
A subset of equine sarcoids harbours BPV-1 DNA in a complex with L1 major capsid protein. Bovine papillomavirus type 1 or 2 (BPV-1, BPV-2) are accepted causal factors in equine sarcoid pathogenesis. Whereas viral genomes are consistently found and expressed within lesions, intact virions have never been detected, thus permissiveness of sarcoids for BPV-1 replication remains unclear. To reassess this issue, an immunocapture PCR (IC/PCR) was established using L1-specific antibodies to capture L1-DNA complexes followed by amplification of the viral genome. Following validation of the assay, 13 sarcoid-bearing horses were evaluated by IC/PCR. Samples were derived from 21 tumours, 4 per...
An equine chromosome 3 inversion is associated with the tobiano spotting pattern in German horse breeds. The tobiano white-spotting pattern is one of several known depigmentation phenotypes in horses and is desired by many horse breeders and owners. The tobiano spotting phenotype is inherited as an autosomal dominant trait. Horses that are heterozygous or homozygous for the tobiano allele (To) are phenotypically indistinguishable. A SNP associated with To had previously been identified in intron 13 of the equine KIT gene and was used for an indirect gene test. The test was useful in several horse breeds. However, genotyping this sequence variant in the Lewitzer horse breed revealed that 14% of ho...
Lactobacillus equigenerosi sp. nov., a coccoid species isolated from faeces of thoroughbred racehorses. Two strains of lactic acid bacteria were isolated from faeces of two actively racing thoroughbred horses. The isolates formed a subcluster in the Lactobacillus reuteri phylogenetic group, closely related to Lactobacillus fermentum, L. gastricus, L. ingluviei and L. mucosae, by phylogenetic analysis based on 16S rRNA gene sequences. Levels of DNA-DNA relatedness revealed that the isolates belonged to the same taxon and were genetically separated from their phylogenetic relatives. Biochemical and physiological characteristics also distinguished the isolates from their phylogenetic relatives. The...
Ancient DNA clarifies the evolutionary history of American Late Pleistocene equids. Hippidions are past members of the equid lineage which appeared in the South American fossil record around 2.5 Ma but then became extinct during the great late Pleistocene megafaunal extinction. According to fossil records and numerous dental, cranial, and postcranial characters, Hippidion and Equus lineages were expected to cluster in two distinct phylogenetic groups that diverged at least 10 MY, long before the emergence of the first Equus. However, the first DNA sequence information retrieved from Hippidion fossils supported a striking different phylogeny, with hippidions nesting inside a p...
Chimeric vapA/groEL2 DNA vaccines enhance clearance of Rhodococcus equi in aerosol challenged C3H/He mice. Rhodococcus equi remains a significant bacterial pathogen, causing severe pyogranulomatous pneumonia in foals aged 1-3 months. There is no effective vaccine currently available for the prevention of R. equi pneumonia. DNA vaccines are known to offer specific advantages over conventional vaccines. The aim of this study was to demonstrate efficacy of our recombinant DNA vaccine candidates, namely pcDNA3-Re1, pcDNA3-Re3 and pcDNA3-Re5 by combining a heat shock protein GroEL2 to a virulence-associated protein A (VapA) from R. equi to protect C3H/He mice against the R. equi infection. VapA was show...
Semi-quantitative analysis of Ruminococcus flavefaciens, Fibrobacter succinogenes and Streptococcus bovis in the equine large intestine using real-time polymerase chain reaction. There is a need to further our understanding of the role that the equine hindgut ecosystem plays in digestive processes and diseases. The aim of the present study was to utilise the real-time PCR technique to determine the abundance of candidate cellulolytic (Ruminococcus flavefaciens; Fibrobacter succinogenes) and non-cellulolytic (Streptococcus bovis) bacteria in lumen contents from the caecum, ventral and dorsal colon, and rectum of healthy horses (n 14). Total DNA was extracted from frozen and lyophilised lumen contents, and PCR primers and Taqman probes were designed based on 16S rDNA seq...
Molecular cloning and characterization of equine Toll-like receptor 9. Innate immunity relies on a series of germline-encoded pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), to detect conserved microbial components. TLR9 is typically expressed intracellularly in immune cells such as dendritic cells and recognizes unmethylated bacterial or viral cytosine-phosphate-guanine DNA (CpG-DNA). To investigate innate immune responses through TLR9 signaling pathway in horses, we cloned and characterized equine TLR9. Protein sequence analysis shows that equine TLR9 has a typically conserved cytosolic Toll/interleukin-1 receptor (TIR) domain, three l...
Glycogen synthase (GYS1) mutation causes a novel skeletal muscle glycogenosis. Polysaccharide storage myopathy (PSSM) is a novel glycogenosis in horses characterized by abnormal glycogen accumulation in skeletal muscle and muscle damage with exertion. It is unlike glycogen storage diseases resulting from known defects in glycogenolysis, glycolysis, and glycogen synthesis that have been described in humans and domestic animals. A genome-wide association identified GYS1, encoding skeletal muscle glycogen synthase (GS), as a candidate gene for PSSM. DNA sequence analysis revealed a mutation resulting in an arginine-to-histidine substitution in a highly conserved region of G...
