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.
Feofilov AV, Bardukov NV, Glazko VI.Using ISSR-PCR marker data, comparative analysis of the gene pools of Altaic and trotting horse breeds was carried out. Horse groups of different origin demonstrated differences in amplification spectra of DNA fragments flanked by inverted repeats of four microsatellites. Combinations of certain DNA fragments present in these profiles reproducibly distinguished genomes of the Altaic breed from the trotting breeds. Genetic differentiation between some trotting breeds, based on Nei genetic distance values, was found to be comparable to that between the groups of horses of Altaic breed from two d...
Leon A, Richard E, Fortier C, Laugier C, Fortier G, Pronost S.Abortion, stillbirth and neonatal death are major causes of equine mortality and cause severe economic loss to the equine industry. The present study was based on a complete necropsy protocol associated with classical microbiological examinations and molecular biology on 407 cases of abortion, stillbirths and neonate death. Based on this retrospective survey, "less common" abortive infectious agents were characterised by molecular tools in nine independent cases of abortion or neonate mortality. Among others, Chlamydophila abortus (1 case), Coxiella burnetii (6 cases) and Neospora caninum (3 c...
Edmond AJ, Brinsko SP, Love CC, Blanchard TL, Teague SR, Varner DD.Centrifugal fractionation of semen is commonly done to improve quality of human semen in assisted-reproduction laboratories, allowing sperm separation based on their isopycnic points. Sperm with morphologic abnormalities are often more buoyant, promoting their retention above defined density media, with structurally normal sperm passing through the media following centrifugation. Three experiments were conducted to evaluate the effects of density-medium type, centrifuge-tube size, sperm number, and density-medium volume (column height) on stallion sperm quality and recovery rate in sperm pelle...
Révay T, Villagómez DA, Brewer D, Chenier T, King WA.Genetic sex in mammals is determined by the sex chromosomal composition of the zygote. The X and Y chromosomes are responsible for numerous factors that must work in close concert for the proper development of a healthy sexual phenotype. The role of androgens in case of XY chromosomal constitution is crucial for normal male sex differentiation. The intracellular androgenic action is mediated by the androgen receptor (AR), and its impaired function leads to a myriad of syndromes with severe clinical consequences, most notably androgen insensitivity syndrome and prostate cancer. In this paper, w...
Lippold S, Matzke NJ, Reissmann M, Hofreiter M.DNA target enrichment by micro-array capture combined with high throughput sequencing technologies provides the possibility to obtain large amounts of sequence data (e.g. whole mitochondrial DNA genomes) from multiple individuals at relatively low costs. Previously, whole mitochondrial genome data for domestic horses (Equus caballus) were limited to only a few specimens and only short parts of the mtDNA genome (especially the hypervariable region) were investigated for larger sample sets. Results: In this study we investigated whole mitochondrial genomes of 59 domestic horses from 44 breeds an...
Sun X, Yao H, Zhang C, Lu C.Equine herpesvirus type 1 (EHV-1) is a major cause of respiratory and reproductive diseases in horses worldwide. The genome of EHV-1 strain 438/77 (isolated from an aborted equine fetus) was cloned as a bacterial artificial chromosome (BAC) in E. coli without any gene deletions. The mini-F plasmid sequence was inserted in the middle of ORF19 and 20 via homologous recombination following co-transfection of viral DNA and plasmid pE19_20/HA into RK13 cells. Circular viral DNA was extracted from RK13 cells infected with purified recombinant virus expressing green fluorescent protein (GFP) and elec...
Binns MM, Boehler DA, Bailey E, Lear TL, Cardwell JM, Lambert DH.Changes in the inbreeding coefficient, F, in the Thoroughbred horse over the past 45 years have been investigated by genotyping 467 Thoroughbred horses (born between 1961 and 2006) using the Illumina Equine SNP50 bead chip, which comprises 54,602 SNPs uniformly distributed across the equine genome. The Spearman rank correlation coefficient, r, between the year of birth and F was estimated. The results indicate that inbreeding in Thoroughbreds has increased over the past 40 years, with r = 0.24, P < 0.001 demonstrating that there is a highly significant, though relatively weak correlation be...
Wnuk M, Villagómez DA, Bugno-Poniewierska M, Tumidajewicz P, Carter TF, Slota E.Nucleolar organizing regions (NORs) containing rDNA gene clusters have been assigned to the equine autosomes ECA1, ECA28, and ECA31. Active NORs (Ag-NORs) are associated with argyrophilic proteins, which allow them to be readily identified using silver staining techniques. Fluorescence in situ hybridization (FISH) for rDNA can also be used to visualize all NOR clusters in the nucleus, regardless of whether they are active or inactive. The present study analyzed the distribution and behavior of equine Ag-NOR and NOR clusters in horse spermatozoa and during male meiosis by FISH and silver staini...
Müller JM, Wissemann J, Meli ML, Dasen G, Lutz H, Heinzerling L, Feige K.Whole blood pharmacokinetics of intratumourally injected naked plasmid DNA coding for equine Interleukin 12 (IL-12) was assessed as a means of in vivo gene transfer in the treatment of melanoma in grey horses. The expression of induced interferon gamma (IFN-g) was evaluated in order to determine the pharmacodynamic properties of in vivo gene transduction. Seven grey horses bearing melanoma were injected intratumourally with 250 µg naked plasmid DNA coding for IL-12. Peripheral blood and biopsies from the injection site were taken at 13 time points until day 14 post injection (p.i.). Samples w...
Shepherd ML, Swecker WS, Jensen RV, Ponder MA.The diversity of the equine fecal bacterial community was evaluated using pyrosequencing of 16S rRNA gene amplicons. Fecal samples were obtained from horses fed cool-season grass hay. Fecal bacteria were characterized by amplifying the V4 region of bacterial 16S rRNA gene. Of 5898 mean unique sequences, a mean of 1510 operational taxonomic units were identified in the four fecal samples. Equine fecal bacterial richness was higher than that reported in humans, but lower than that reported in either cattle feces or soil. Bacterial classified sequences were assigned to 16 phyla, of which 10 were ...
Georgescu SE, Manea MA, Dudu A, Costache M.The existence of the Hucul horse on Romanian territory has been documented from the very distant past; today Hucul is a unique breed that is part of the FAO Program for the Preservation of Animal Genetic Resources. We compared Hucul with several primitive European and Asiatic breeds in order to elucidate the origin of these horses. We analyzed a 683-bp mitochondrial DNA (mtDNA) D-loop fragment in a population of Hucul horses and compared the polymorphic sites with sequences from other primitive breeds, including Exmoor, Icelandic Pony, Sorraia, Przewalski Horse, Mongolian Wild Horse, Konik, an...
Ciotola F, Albarella S, Pasolini MP, Auletta L, Esposito L, Iannuzzi L, Peretti V.An 18-month-old Arabian foal characterized by a stallion-like appearance was submitted for cytogenetic and molecular genetics examinations due to abnormalities of external genitalia and the presence of ovotestis-like structures in the abdominal cavity. By RB-banding the animal showed the normal female equine karyotype (2n = 64,XX). Molecular analysis revealed the absence of the SRY and ZFY genes and the presence of ZFX, a typical female equine condition. The entire RSPO1 coding region was examined to exclude its involvement. Although a SNP was found in exon 3, it was not responsible for an ami...
Campana MG, Stock F, Barrett E, Benecke N, Barker GW, Seetah K, Bower MA.Despite the Icelandic horse enjoying great popularity worldwide, the breed's gene pool is small. This is because of a millennium of isolation on Iceland, population crashes caused by natural disasters and selective breeding. Populations with small effective population sizes are considered to be more at risk of selection pressures such as disease and environmental change. By analysing historic and modern mitochondrial DNA sequences and nuclear coat colour genes, we examined real-time population dynamics in the Icelandic horse over the last 150 years. Despite the small gene pool of this breed, w...
de Leon PM, Campos VF, Dellagostin OA, Deschamps JC, Seixas FK, Collares T.In this study, polymerase chain reaction (PCR) reamplification of the first PCR product (2nd-PCR) and a qPCR assay were used to detect the sex determining region Y (SRY) gene from circulating cell-free fetal DNA (ccffDNA) in blood plasma of pregnant mares to determine fetal sex. The ccffDNA was isolated from plasma of 20 Thoroughbred mares (5-13 y old) in the final 3 mo of pregnancy (fetal sex was verified after foaling). For controls, plasma from two non-pregnant mares and two virgin mares were used, in addition to the non-template control. The 182 bp nucleotide sequence corresponding to the ...
Cothran EG, Canelon JL, Luis C, Conant E, Juras R.Various horse populations in the Americas have an origin in Spain; they are remnants of the first livestock introduced to the continent early in the colonial period (16th and 17th centuries). We evaluated genetic variability within the Venezuelan Criollo horse and its relationship with other horse breeds. We observed high levels of genetic diversity within the Criollo breed. Significant population differentiation was observed between all South American breeds. The Venezuelan Criollo horse showed high levels of genetic diversity, and from a conservation standpoint, there is no immediate danger ...
Mata X, Vaiman A, Ducasse A, Diribarne M, Schibler L, Guérin G.Gene characterization is an important feature for genome annotation and more particularly for candidate genes that could be selected in domestic species. Associations between an alpha-actinin-3 gene polymorphism and muscle performance were reported in humans involving a nonsense mutation (R577X) and in mice after inactivation of the gene. Here, we characterized the equine alpha-actinin-3 (ACTN3) gene by sequencing and transcript analysis. The cDNA was determined to be 3.47 kb in length with an open reading frame of 2709 bp expectedly encoding a protein 902 amino acids long. The ACTN3 gene is 1...
Herder V, Barsnick R, Walliser U, Teifke JP, König P, Czerwinski G, Hansmann F, Baumgärtner W, Hewicker-Trautwein M.An equid herpesvirus 5 (EHV-5) infection was detected in lesioned skin from a nine-year-old Holsteiner stallion in the south of Germany. Macroscopically, the animal displayed a non-pruritic, multifocal, pustular dermatitis around both eyes, nostrils and the muzzle, which had been ongoing for one year. Histopathologically, skin lesions were characterized by orthokeratotic to parakeratotic hyperkeratosis, pustular dermatitis, epidermal hyperplasia, apoptotic keratinocytes, a lympho-plasmahistiocytic interface dermatitis with hydropic degeneration of keratinocytes, and perivascular to diffuse, ly...
Smits K, Govaere J, Hoogewijs M, Piepers S, Van Soom A.Intracytoplasmic sperm injection (ICSI) is the method of choice for the in vitro production (IVP) of equine embryos. However, conventional ICSI has been associated with mechanical damage to the oocyte caused by the deformation of the zona pellucida (ZP) and exposure of the oolemma to negative pressure during injection. Introduction of the less traumatic and more efficient piezo drill-assisted ICSI (PDAI) yielded higher cleavage rates and more consistent results. Nevertheless, PDAI is also associated with disadvantages such as the use of mercury and possible DNA damage. This led us to explore a...
Böer U, Lohrenz A, Klingenberg M, Pich A, Haverich A, Wilhelmi M.Decellularized equine carotid arteries (dEAC) may represent a reasonable alternative to alloplastic materials in vascular replacement therapy. Acellularity of the matrix is standardly evaluated by DNA quantification what however may not record sufficiently the degree of matrix immunogenicity. Thus, our aim was to analyze dEAC with a low DNA content for residual cellular proteins. A detergent-based decellularization protocol including endonuclease treatment resulted in dEAC with 0.6 ± 0.15 ng DNA/mg dry weight representing 0.33 ± 0.14% of native tissue DNA content. In contrast, when matrices ...
Corbin LJ, Blott SC, Swinburne JE, Vaudin M, Bishop SC, Woolliams JA.We have used linkage disequilibrium (LD) to identify single nucleotide polymorphisms (SNPs) on the Illumina Equine SNP50 BeadChip, which may be incorrectly positioned on the genome map. A total of 1201 Thoroughbred horses were genotyped using the Illumina Equine SNP50 BeadChip. LD was evaluated in a pairwise fashion between all autosomal SNPs, both within and across chromosomes. Filters were then applied to the data, firstly to identify SNPs that may have been mapped to the wrong chromosome and secondly to identify SNPs that may have been incorrectly positioned within chromosomes. We identifie...
The Journal of hereditySeptember 13, 2011
Volume 102, Issue 6 753-758 doi: 10.1093/jhered/esr091
Guastella AM, Zuccaro A, Criscione A, Marletta D, Bordonaro S.Genetic diversity and relationship among 3 Sicilian horse breeds were investigated using 16 microsatellite markers and a 397-bp length mitochondrial D-loop sequence. The analysis of autosomal DNA was performed on 191 horses (80 Siciliano [SIC], 61 Sanfratellano [SAN], and 50 Sicilian Oriental Purebred [SOP]). SIC and SAN breeds were notably higher in genetic variability than the SOP. Genetic distances and cluster analysis showed a close relationship between SIC and SAN breeds, as expected according to the breeds' history. Sequencing of hypervariable mitochondrial DNA region was performed on a ...
Artiushin S, Tong Y, Timoney J, Lemieux B, Schlegel A, Kong H.A simple and portable assay for detection of Streptococcus equi subspecies equi has been developed based on amplification of S. equi-specific sequence using a thermophilic helicase-dependent reaction followed by visual detection of the amplicon in a disposable lateral flow cassette. An experimental kit (IsoAmp™ SE) was evaluated. Analytical sensitivity was 50 copies of S. equi genomic DNA per reaction. The IsoAmp SE assay had 100% specificity when applied to nasal swabs and washes. The assay was more sensitive than culture but less sensitive than nested polymerase chain reaction (PCR). The t...
Bae JH, Ahn K, Nam GH, Lee CE, Park KD, Lee HK, Cho BW, Kim HS.The horse BMAL1 gene encodes the brain and muscle Arnt-like protein 1, which is a key regulator of circadian rhythmic systems in most organs and cells. The first exon of the horse-specific BMAL1 gene is produced by an exonization event of LINE3 (CR1) and SINE (MIR) was detected by bioinformatic analysis. Alternative variants generated by cassette exon event in various horse tissues were also detected by RT-PCR amplification and sequencing. The cDNA sequences of the horse transcripts (BMAL1a, BMAL1b) contain additional 21 bp and 71 bp fragments relative to horse BMAL1. Quantitative real-time RT...
Lippold S, Knapp M, Kuznetsova T, Leonard JA, Benecke N, Ludwig A, Rasmussen M, Cooper A, Weinstock J, Willerslev E, Shapiro B, Hofreiter M.Modern domestic horses display abundant genetic diversity within female-inherited mitochondrial DNA, but practically no sequence diversity on the male-inherited Y chromosome. Several hypotheses have been proposed to explain this discrepancy, but can only be tested through knowledge of the diversity in both the ancestral (pre-domestication) maternal and paternal lineages. As wild horses are practically extinct, ancient DNA studies offer the only means to assess this ancestral diversity. Here we show considerable ancestral diversity in ancient male horses by sequencing 4 kb of Y chromosomal DNA ...
Silaghi C, Liebisch G, Pfister K.Equine Granulocytic Anaplasmosis (EGA) is caused by Anaplasma phagocytophilum, a tick-transmitted, obligate intracellular bacterium. In Europe, it is transmitted by Ixodes ricinus. A large number of genetic variants of A. phagocytophilum circulate in nature and have been found in ticks and different animals. Attempts have been made to assign certain genetic variants to certain host species or pathologies, but have not been successful so far. The purpose of this study was to investigate the causing agent A. phagocytophilum of 14 cases of EGA in naturally infected horses with molecular methods o...
Tsujimura K, Oyama T, Katayama Y, Muranaka M, Bannai H, Nemoto M, Yamanaka T, Kondo T, Kato M, Matsumura T.A single non-synonymous nucleotide substitution of guanine (G) for adenine (A) at position 2254 in the viral DNA polymerase gene (encoded by open reading frame [ORF] 30) of equine herpesvirus type 1 (EHV-1) has been significantly associated with neuropathogenic potential in strains of this virus. To estimate the prevalence of EHV-1 strains with the neuropathogenic genotype (ORF30 G(2254)) in the Hidaka district--a major horse breeding area in Japan--we analyzed the ORF30 genomic region in cases of EHV-1 infection in this area during the years 2001-2010. Of the 113 cases analyzed, 3 (2.7%) were...
Orlando L, Ginolhac A, Raghavan M, Vilstrup J, Rasmussen M, Magnussen K, Steinmann KE, Kapranov P, Thompson JF, Zazula G, Froese D, Moltke I....Second-generation sequencing platforms have revolutionized the field of ancient DNA, opening access to complete genomes of past individuals and extinct species. However, these platforms are dependent on library construction and amplification steps that may result in sequences that do not reflect the original DNA template composition. This is particularly true for ancient DNA, where templates have undergone extensive damage post-mortem. Here, we report the results of the first "true single molecule sequencing" of ancient DNA. We generated 115.9 Mb and 76.9 Mb of DNA sequences from a permafrost-...
Goto H, Ryder OA, Fisher AR, Schultz B, Kosakovsky Pond SL, Nekrutenko A, Makova KD.The endangered Przewalski's horse is the closest relative of the domestic horse and is the only true wild horse species surviving today. The question of whether Przewalski's horse is the direct progenitor of domestic horse has been hotly debated. Studies of DNA diversity within Przewalski's horses have been sparse but are urgently needed to ensure their successful reintroduction to the wild. In an attempt to resolve the controversy surrounding the phylogenetic position and genetic diversity of Przewalski's horses, we used massively parallel sequencing technology to decipher the complete mitoch...
Kim SK, Kim S, Dai G, Zhang Y, Ahn BC, O'Callaghan DJ.The equine herpesvirus 1 (EHV-1) negative regulatory IR2 protein (IR2P), an early 1,165-amino acid (aa) truncated form of the 1487-aa immediate-early protein (IEP), lacks the trans-activation domain essential for IEP activation functions but retains domains for binding DNA, TFIIB, and TBP and the nuclear localization signal. IR2P mutants of the N-terminal region which lack either DNA-binding activity or TFIIB-binding activity were unable to down-regulate EHV-1 promoters. In EHV-1-infected cells expressing full-length IR2P, transcription and protein expression of viral regulatory IE, early EICP...
Wang X, Bou G, Zhang X, Tao L, Shen Y, Na R, Liu G, Ren H, Ren X, Song L, Su S, Bai D, Zhao Y, Li B, Dugarjaviin M.Having considered that the current methods are costly and time-consuming, we designed an only 3 pairs primer-based PCR test to accurately identify the species and gender in horses, donkeys, mules and hinnies. Through a thorough sequence comparison between horse and donkey's highly similar genomes, and a vast amount of preliminary confirmation, we found that three fragments, CNGB3 gene on an autosome, displacement loop region on mitochondrial DNA and SRY genes on chromosome Y, within these equine DNA, are enough to enable us achieving our goal. The PCR test described here would be an economical...
Drebert Z, Golke A, Cymerys J, Słońska A, Chmielewska A, Tucholska A, Bańbura MW.Equid herpesvirus type 1 (EHV-1) is a prevalent causative agent of equine diseases worldwide. After primary replication in the respiratory epithelium the virus disseminates systemically through a peripheral blood mononuclear cell (PBMC)-associated viraemia. EHV-1 is the only alphaherpes- virus known so far which is capable of establishing latent infection not only in neurons but also in immune system cells (mainly in lymphocytes and macrophages). Since leukocytes are not the target cells for viral replication but are used to transport EHV-1 to the internal organs, the questionremains how the v...
Drogemuller M, Schnieder T, von Samson-Himmelstjerna G.P-glycoproteins (Pgps) are adenosine triphosphate-binding transporter proteins thought to be associated with multi-drug resistance in mammals and protozoans and have been suggested to be involved in the mechanism of ivermectin (IVM) resistance in Haemonchus contortus. Until now, resistance to IVM has not been reported in cyathostomins in horses in spite of its widespread and frequent use. Reasons for this might be differences in the molecular mechanism of the development of resistance. Based on this hypothesis, the present study was carried out to find homologues of Pgp in cyathostomins. A 416...
Hinshelwood MM, Liu Z, Conley AJ, Simpson ER.Conversion of androgens to estrogens is catalyzed by aromatase P450 (P450arom; the product of the CYP19 gene). Regulation of tissue-specific expression of P450arom in humans is due, in part, to alternative transcriptional start sites that arise as a consequence of the use of granulosa cells and placental tissue from cows, horses, and pigs (ungulates) in order to determine whether these species, like the human, utilize tissue-specific promoters to drive P450arom expression. The majority of transcripts in the placenta have 5'-termini that differ from those in the ovary upstream of a common site ...
Carvalho R, Passos LM, Martins AS.In this study, a multiplex polymerase chain reaction (PCR) procedure was developed for differentiation of strains and field isolates of equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4). Specific oli-gonucleotide primers were combined to amplify the thymidine kinase (TK) gene region of EHV-1 and EHV-4, which would yield fragments of different lengths for each virus in the same amplification reaction. The specificity of the largest PCR amplicon for EHV-4 was confirmed by restriction digestion with HindIII. The multiplex PCR proved to be a fast and sensitive method for typing EHV-1 and EHV-4 ...
RANDALL CC, WALKER BM.Randall, Charles C. (University of Mississippi School of Medicine, Jackson) and Barbara M. Walker. Degradation of deoxyribonucleic acid and alteration of nucleic acid metabolism in suspension cultures of L-M cells infected with equine abortion virus. J. Bacteriol. 86:138-146. 1963.-Metabolic alterations in log-phase suspension cultures infected with equine abortion virus (EAV) were determined in L-M cells simultaneously labeled or prelabeled with H(3)- or C(14)-thymidine. Although infection produced an early stimulation of the uptake of labeled thymidine (TdR) into the acid-soluble fraction of...
Wierdl M, Morton CL, Danks MK, Potter PM.Butyrylcholinesterases (BuChEs; acylcholine acylhydrolase; EC 3.1.1.8) have been demonstrated to convert the anticancer agent CPT-11 (irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) into its active metabolite SN-38 (7-ethyl-10-hydroxycamptothecin). In addition, significant differences in the extent of drug metabolism have been observed with BuChEs derived from different species. In an attempt to understand these differences, we have isolated the cDNA encoding a horse BuChE. Based upon the NH2-terminal amino acid sequence of a purified horse BuChE, we designed deg...
Carpenter S, Baker JM, Bacon SJ, Hopman T, Maher J, Ellis SA, Antczak DF.Sequence and functional analyses were undertaken on two cDNAs and a genomic clone encoding horse major histocompatibility complex (MHC) class I molecules. All of the clones were isolated from a single horse that is homozygous for all known horse MHC class I and class II antigens. The two cDNAs (clones 8-9 and 1-29) were isolated from a lymphocyte library and encode polymorphic MHC antigens from two loci. The genomic cosmid clone, isolated from a sperm library, contains the 8-9 gene. All three genes were expressed in mouse L-cells and were recognized by alloantisera and, for the cDNAs, by allor...
Demyda-Peyrás S, Membrillo A, Bugno-Poniewierska M, Pawlina K, Anaya G, Moreno-Millán M.Chromosomal abnormalities associated to sex chromosomes are reported as a problem more common than believed to be in horses. Most of them remain undiagnosed due to the complexity of the horse karyotype and the lack of interest of breeders and veterinarians in this type of diagnosis. Approximately 10 years ago, the Spanish Purebred Breeders Association implemented a DNA paternity test to evaluate the pedigree of every newborn foal. All candidates who showed abnormal or uncertain results are routinely submitted to cytogenetical analysis to evaluate the presence of chromosomal abnormalities. We s...
Lindquist CD, Evans JJ, Wictum EJ.Accurate DNA quantification is essential for optimizing DNA testing and minimizing sample consumption. Real-time quantitative polymerase chain reaction (qPCR) assays have been published for human and canine nuclear DNA, and the need for quantifying other forensically important species was evident. Following the strategy employed for the canine qPCR assay, we developed individual assays to accurately quantify feline, bovine, equine, and cervid nuclear DNA. Each TaqMan-based assay incorporates a genus-specific probe targeting the Melanocortin-1 Receptor gene and includes a piece of synthetic DNA...
Ayoub C, Arroyo LG, Renaud D, Weese JS, Gomez DE.The objective of this study was to compare the fecal microbiota of two healthy teaching horse herds with that of client-owned horses from the same geographic areas. The fecal microbiota of client-owned horses from Ontario Canada (n = 15) and Florida, USA (n = 11) was compared with that teaching horses from the University of Guelph, Ontario, Canada (n = 10) and the University of Florida, Florida, USA (n = 15). The fecal microbiota was characterized by sequencing of bacterial DNA using the V4 hypervariable region of the 16S rRNA gene. The diversity (inverse Simpson index) of the fecal mi...
Lepage N, St-Laurent G, Carman S, Archambault D.The genetic variation in equine arteritis virus (EAV) Gs protein encoding gene was investigated. Nucleic and deduced amino acid sequences from eight different EAV isolates (one European, two American and five Canadian isolates) were compared with those of the Bucyrus reference strain. Nucleotide and amino acid identities between these isolates and the Bucyrus reference strain ranged from 92.3 to 96.4%, and 93.2 to 95.5%, respectively. However, phylogenetic tree analysis and estimation of genetic distances based on the Gs protein encoding gene sequences showed that the European prototype Vienna...
Muscatello G, Browning GF.Selective agar media have been used for many years to facilitate the isolation of Rhodococcus equi from environmental and clinical samples. However, characterisation of R. equi still requires the use of immunochemical or polymerase chain reaction (PCR) analysis to differentiate between virulent and avirulent isolates. Here, we describe a novel method to detect and differentiate between R. equi isolates using colony blotting and DNA hybridization. Radiolabelled PCR product derived from the R. equi rrnA gene and specific hybridization conditions enabled differentiation of colonies of R. equi fro...
Nasir L, Gault E, Morgan IM, Chambers G, Ellsmore V, Campo MS.BPV-1 DNA is the predominant viral type detected in equine sarcoids and represents the only reported natural cross species infection of papillomaviruses. In this study, nucleotide variations in the LCR and the E2 regions of equine sarcoid-associated BPV-1 were characterised by sequence analysis. Variants particular to sarcoid BPV-1 were identified in both the LCR and E2 sequence. The functionality of the most common LCR variant was examined in equine and bovine cells. These studies showed that the activity of the variant LCR was higher in equine cells than bovine cells; the activity of the var...
von Samson-Himmelstjerna G, Janssen IJI, Ramünke S, Goday C, Borges FA, Koudela B, Niedźwiedź A, Tomczuk K, Studzińska MB, Kornas S, Krücken J.Equines were over decades considered to be infected by two morphologically virtually indistinguishable ascarid species, Parascaris univalens and Parascaris equorum. Reliable species discrimination is only possible using enzyme isoelectric focussing and karyotyping with P. univalens having one and P. equorum two chromosome pairs. However, presumably the complexity of both methods prevented their routine use in nearly all previous studies about prevalence and drug resistance of Parascaris spp. These have barely been performed on the species level although most studies stated presence of one or t...
Oldenhof H, Schütze S, Wolkers WF, Sieme H.Sperm chromatin structure and condensation determine accessibility for damage, and hence success of fertilization and development. The aim of this study was to reveal characteristic spectral features coinciding with abnormal sperm chromatin packing (i.e., DNA-protein interactions) and decreased fertility, using Fourier transform infrared spectroscopy. Chromatin structure in spermatozoa obtained from different stallions was investigated. Furthermore, spermatozoa were exposed to oxidative stress, or treated with thiol-oxidizing and disulfide-reducing agents, to alter chromatin structure and pack...
Smith KC, Borchers K.The polymerase chain reaction and DNA in-situ hybridization were used to study sections of uterine tissue collected from mares near the time of abortion due to equid herpesvirus-1 (EHV-1) infection. These techniques revealed viral nucleic acids in endothelial cells of endometrial arterioles, in accordance with previously published immunohistological data. In addition, however, they revealed nucleic acids in cellular debris within endometrial glands and diffusing across the placenta at sites of microcotyledonary infarction. Perivascular leucocytes were generally negative for viral DNA, despite ...
Savill MG, Murray SR, Scholes P, Maas EW, McCormick RE, Moore EB, Gilpin BJ.Rhodococcus coprophilus, a natural inhabitant of herbivore faeces, has been suggested as a good indicator of animal (as opposed to human) faecal contamination of aquatic environments. However, conventional detection methods limit its use for this as they require up to 21 days to obtain a result. In this paper an optimised method for extracting R. coprophilus DNA from faecal samples is described. PCR and 5'-nuclease (TaqMan) PCR methods were developed to allow the detection and enumeration of R. coprophilus in faecal samples within 2-3 days. Both PCR methods targeted the 16S rRNA gene, producin...
Looft C, Paul S, Philipp U, Regenhard P, Kuiper H, Distl O, Chowdhary BP, Leeb T.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...
Phumoonna T, Barton MD, Vanniasinkam T, Heuzenroeder MW.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...
Adaszek Ł, Winiarczyk S, Łukaszewska J.The study was aimed at determining the cause of a disease in a horse exhibiting symptoms of fever, joint effusion, weakness, and extravasations on the mucous membranes. Blood was drawn from the animal for haematological and biochemical molecular tests. The PCR technique revealed the presence of 16S RNA Ehrlichia spp. genetic material in the blood samples. DNA amplification by means of primers EHR 521 and EHR 747 gave a product with a volume of 247 bp.The sequence of the PCR product obtained showed a 97.6% similarity with a sequence of a fragment of 16S RNA Ehrlichia phagocytophila, gene number...
Studdert MJ, Crabb BS, Ficorilli N.The restriction endonuclease DNA fingerprints of 57 isolates of equine herpesvirus 1 (EHV1; equine abortion virus) from abortion, perinatal foal mortalities and encephalitis from 15 epidemics that occurred in Australasia between 1975 and 1989 were examined using the enzymes Bam HI, EcoRI and Bgl II. There was a remarkable degree of uniformity in the restriction patterns; mobility differences were observed in only 14 of 52 (27%) of the fragments. Twelve of these 14 fragments were located within the repeat structures that bracket the unique short region of the genome or were located at the left ...
Borchers K, Frölich K, Ludwig H.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.
Wilkin T, Hamilton NA, Cawley AT, Bhat S, Baoutina A.The term 'gene doping' is used to describe the use of any unauthorized gene therapy techniques. We developed a test for five likely candidate genes for equine gene doping: , , , and . The test is based on real-time polymerase chain reaction (PCR) and includes separate screening and confirmation assays that detect different unique targets in each transgene. For doping material, we used nonviral (plasmid) and viral (recombinant adeno-associated virus) vectors carrying complementary DNA for the targeted genes; the vectors were accurately quantified by digital PCR. To reduce non-specific amplific...
Flack N, Hughes L, Cassens J, Enriquez M, Gebeyehu S, Alshagawi M, Hatfield J, Kauffman A, Brown B, Klaeui C, Mabrouk IF, Walls C, Yeater T, Rivas A....The Przewalski's horse (Equus ferus przewalskii) is an endangered equid native to the steppes of central Asia. After becoming extinct in the wild multiple conservation efforts convened to preserve the species, including captive breeding programs, reintroduction and monitoring systems, protected lands, and cloning. Availability of a highly contiguous reference genome is essential to support these continued efforts. We used Oxford Nanopore sequencing to produce a scaffold-level 2.5 Gb nuclear assembly and 16,002 bp mitogenome from a captive Przewalski's mare. All assembly drafts were generated f...
Diekmann I, Krücken J, Kuzmina TA, Bredtmann CM, Louro M, Kharchenko VA, Tzelos T, Matthews JB, Madeira de Carvalho LM, von Samson-Himmelstjerna G.Morphologically, 64 strongylid species have been described in equines. Co-infections are common, with up to 29 species reported in a single horse. Morphological identification of these species is time consuming and requires expert knowledge due to their similar appearance. Therefore, non-invasive identification methods are needed. DNA barcoding offers a rapid and reliable tool for species identification and the discovery of cryptic species for these most common parasitic nematodes of equines. In total, 269 COI and 312 ITS-2 sequences from 27 equine Strongylidae species, including sequences fro...
Ahedor B, Otgonsuren D, Zhyldyz A, Guswanto A, Ngigi NMM, Valinotti MFR, Kothalawala H, Kalaichelvan N, Silva SSP, Kothalawala H, Acosta TJ....Theileria equi causes equine piroplasmosis, an economically significant disease that affects horses and other equids worldwide. Based on 18S ribosomal RNA (18S rRNA sequences), T. equi can be classified into five genotypes: A, B, C, D, and E. These genotypes have implications for disease management and control. However, no conventional polymerase chain reaction (PCR) assays are available to differentiate the genotypes of T. equi. To overcome this limitation, we developed and evaluated PCR assays specific for the detection of each T. equi genotype. Methods: A pair of forward and reverse primers...
Bull KE, Hodgkinson J, Allen K, Poissant J, Peachey LE.Cyathostomins are the most abundant equid endoparasites globally. There are approximately fifty cyathostomin species and, whilst they occupy distinct niches within the large intestine, they are generally considered to share similar characteristics in terms of pathogenicity and response to drug treatment. There are three classes of anthelmintic licensed in the UK to treat cyathostomins (benzimidazoles, tetrahydropyrimidines and macrocyclic lactones) and cases of resistance have been documented for all classes. Previously, faecal egg count reduction tests (FECRT) on four UK Thoroughbred studs re...
Musiał AD, Radović L, Stefaniuk-Szmukier M, Bieniek A, Wallner B, Ropka-Molik K.Polish Konik remains one of the most important horse breeds in Poland. The primitive, native horses with a stocky body and mouse-like coat color are protected by a conservation program, while their Polish population consists of about 3,480 individuals, representing 16 dam and six sire lines. To define the population's genetic structure, mitochondrial DNA and Y chromosome sequence variables were identified. The mtDNA whole hypervariable region analysis was carried out using the Sanger sequencing method on 233 Polish Koniks belonging to all dam lines, while the Y chromosome analysis was performe...
While CENP-A is the epigenetic determinant of the centromeric function, the role of CENP-B, a centromeric protein binding a specific DNA sequence, the CENP-B-box, remains elusive. In the few mammalian species analyzed so far, the CENP-B box is contained in the major satellite repeat that is present at all centromeres, with the exception of the Y chromosome. We previously demonstrated that, in the genus Equus, numerous centromeres lack any satellite repeat. Results: In four Equus species, CENP-B is expressed but does not bind the majority of satellite-based centromeres, or the satellite-free on...
