In situ hybridization (ISH) is a molecular technique used to detect specific nucleic acid sequences within fixed tissues and cells. In equine research, ISH is utilized to study gene expression patterns and the localization of specific RNA or DNA sequences in horse tissues. This technique provides insights into the spatial and temporal aspects of gene activity, helping to elucidate the molecular mechanisms underlying various physiological and pathological processes in horses. ISH can be applied to a range of tissues, including those affected by diseases, to better understand the genetic contributions to equine health and disease. This page compiles peer-reviewed research studies and scholarly articles that explore the applications, methodologies, and findings of in situ hybridization in equine studies.
Bugno M, Jabłońska Z, Słota E.The aim of the study was to optimize hybridization conditions of molecular probes specific for X sex chromosomes of the domestic horse in mare oocyte chromosomes. Mare oocytes, recovered from slaughterhouse ovaries by scraping the granulosa layer, were cultured in vitro. Metaphase II mature oocytes were treated with hypotonic solution and fixed, followed by hybridization of the molecular probe specific for the X chromosome ofthe domestic horse. Hybridization of probes specific for mouse heterosomes on mouse oocytes and early embryos was performed to verify the FISH technique. Of 438 oocytes an...
Hepperger C, Mayer A, Merz J, Vanderwall DK, Dietzel S.Whether chromosome sets inherited from father and mother occupy separate spaces in the cell nucleus is a question first asked over 110 years ago. Recently, the nuclear organization of the genome has come increasingly into focus as an important level of epigenetic regulation. In this context, it is indispensable to know whether or not parental genomes are spatially separated. Genome separation had been demonstrated for plant hybrids and for the early mammalian embryo. Conclusive studies for somatic mammalian cell nuclei are lacking because homologous chromosomes from the two parents cannot be d...
Haneda S, Nagaoka K, Nambo Y, Kikuchi M, Nakano Y, Matsui M, Miyake Y, Macleod JN, Imakawa K.To identify factors involved in the establishment of pregnancy in the mare, endometrium was collected from day 13 (day 0=day of ovulation) cyclic and day 13, 19, and 25 pregnant animals. From initial cDNA subtraction studies, interleukin-1 receptor antagonist (IL-1RN) mRNA was found as a candidate molecule expressed uniquely in the pregnant endometrium. Expression of IL-1RN mRNA was markedly increased in day 19 and 25 gravid endometrium. In situ hybridization analysis revealed that IL-1RN mRNA was localized to the glandular epithelium. Interleukin-1 receptor antagonist (IL-1RN) protein was fou...
Bugno-Poniewierska M, Jabłońska Z, Słota E.Fluorescence in situ hybridization (FISH) is widely used in the study of chromosome structure and organization. Cytogenetic evaluation of chromosomes using FISH technique plays an increasingly important role in diagnosing karyotype changes in both somatic and reproductive cells. The aim of the study was to optimize the conditions of stallion sperm decondensation, which have a significant effect on the results of fluorescence in situ hybridization. Appropriate type and time of decondensation was chosen for the sperm of every stallion. It was found that decondensation performed using a preparati...
Raudsepp T, Gustafson-Seabury A, Durkin K, Wagner ML, Goh G, Seabury CM, Brinkmeyer-Langford C, Lee EJ, Agarwala R, Stallknecht-Rice E, Schäffer AA....A comprehensive second-generation whole genome radiation hybrid (RH II), cytogenetic and comparative map of the horse genome (2n = 64) has been developed using the 5000rad horse x hamster radiation hybrid panel and fluorescence in situ hybridization (FISH). The map contains 4,103 markers (3,816 RH; 1,144 FISH) assigned to all 31 pairs of autosomes and the X chromosome. The RH maps of individual chromosomes are anchored and oriented using 857 cytogenetic markers. The overall resolution of the map is one marker per 775 kilobase pairs (kb), which represents a more than five-fold improvement over ...
Perelygin AA, Zharkikh AA, Astakhova NM, Lear TL, Brinton MA.Chemokine receptors (CCRs) play an essential role in the initiation of an innate immune host response. Several of these receptors have been shown to modulate the outcome of viral infections. The recent availability of complete genome sequences from a number of species provides a unique opportunity to analyze the evolution of the CCR genes. A phylogenetic analysis revealed that the CCR2 gene evolved in concert with the paralogous CCR5 gene, but not with another paralogous gene, CCR3, in the opossum, platypus, rabbit, guinea pig, cat, and rodent lineages. In addition, evidence of concerted evolu...
Brinkmeyer-Langford C, Raudsepp T, Gustafson-Seabury A, Chowdhary BP.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...
Lear TL, Lundquist J, Zent WW, Fishback WD, Clark A.Repeated early embryonic loss (REEL) represents a considerable economic loss to the horse industry. Mares that experience REEL may be overlooked as potential carriers of a chromosome abnormality. Here we report three different autosomal translocations in Thoroughbred mares presented for chromosome analysis because of REEL. The karyotypes were 64,XX,t(1;21), 64,XX,t(16;22), and 64,XX,t(4;13), respectively. In order to confirm the chromosomes involved in the translocations, to map the breakpoints, and to determine if the translocations were reciprocal, genes surrounding the breakpoints were iden...
Sebastian MM, Stewart I, Williams NM, Poonacha KB, Sells SF, Vickers ML, Harrison LR.Pathological, entomological and avian investigations were conducted during the summer of 2002, in a horse farm that had four cases of West Nile virus (WNV) infection in horses. All the four horses had encephalitis and WNV infection was confirmed by RT-PCR and in situ hybridization procedure. Forty-seven per cent of house sparrows that resided on the farm were tested positive for WNV infection. Mosquitoes (98%Culex pipiens) collected by trapping at the farm, during this period were positive for WNV. The meteorological data for year 2002 were compared to previous 16 years. The precipitation and ...
Wnuk M, Bugno M, Slota E.Recently, molecular techniques have become an indispensable tools for cytogenetic research. Especially, development of in situ techniques made possible detection at the chromosomal level, genes as well as repetitive sequences like telomeres or the DNA component of telomeres. One of these methods is primed in situ DNA synthesis (PRINS) using an oligonucleotide primer complementary to the specific DNA sequence. In this report we described application of PRINS technique with telomere human commercial kit to telomere sequences identification. This commercial kit may be use to visualization of inte...
Bugno M, Słota E.A 5-year-old infertile Hutsul mare was subjected to cytogenetic analysis. Fluorescence in situ hybridisation (FISH) using the equine Xp and Xq chromosome painting probes was carried out on chromosome preparations obtained after blood lymphocyte culture. These probes were generated by chromosome microdissection and a large number of spreads was analysed (525). The karyotype formula of the analysed mare was 64,XX/65,XX+Xp with the ratio of the two lines being 99.4 and 0.6, respectively. The goal of the study was to apply chromosome microdissection and the FISH technique for cytogenetic diagnosti...
Milinovich GJ, Trott DJ, Burrell PC, Croser EL, Al Jassim RA, Morton JM, van Eps AW, Pollitt CC.Carbohydrate-induced laminitis in horses is characterized by marked changes in the composition of the hindgut microbiota, from a predominantly Gram-negative population to one dominated by Gram-positive bacteria. The objective of this study was to monitor changes in the relative abundance of selected hindgut bacteria that have previously been implicated in the pathophysiology of equine laminitis using fluorescence in situ hybridization (FISH). Caecal cannulae were surgically implanted in five Standardbred horses and laminitis induced by oral administration of a bolus dose of oligofructose. Caec...
Nomura M, Hosaka Y, Kasashima Y, Ueda H, Takehana K, Kuwano A, Arai K.The DNA microarray analysis for matrix metalloproteinase (MMP)-related mRNA expression in equine superficial digital flexor tendinitis indicated that mRNA level of MMP-13 was apparently up-regulated in the tendinitis as compared to normal tendon. In situ hybridization also revealed that fibroblastic cells proliferated in the granulation tissue generated in the tendinitis actively expressed MMP-13 mRNA. On the other hand, in normal tendon, a few fibroblastic cells and vascular components lied in the endotenon barely expressed its mRNA, but other cellular components in the tendon bundle were not...
Bugno M, Klukowka-Rötzler J, Słota E, Witarski W, Gerber V, Leeb T.The physical localization of the epidermal growth factor receptor (EGFR) gene was performed on donkey chromosomes. Bacterial artificial chromosome DNA containing the equine EGFR gene was used to map this gene by fluorescent in situ hybridization on donkey metaphase chromosomes. The gene was mapped on donkey 1q21.1 region.
Slota E, Wnuk M, Bugno M, Pienkowska-Schelling A, Schelling C, Bratus A, Kotylak Z.Cytogenetic investigations of the nucleolar-organizing regions (NORs) show that there is variation in the transcriptional activity of rDNA in many organisms. As a consequence, genetic polymorphism of these regions has been detected. The aim of the present study was to evaluate the hypothetic genetic mechanisms determining the NORs polymorphism of the domestic horse chromosomes. Molecular cytogenetic analyses were carried out on Hucul horses and the following techniques were used: fluorescence in situ hybridization (FISH), telomere primed in situ synthesis (PRINS), in situ nick-translation with...
Goodrich LR, Hidaka C, Robbins PD, Evans CH, Nixon AJ.Gene therapy with insulin-like growth factor-1 (IGF-1) increases matrix production and enhances chondrocyte proliferation and survival in vitro. The purpose of this study was to determine whether arthroscopically-grafted chondrocytes genetically modified by an adenovirus vector encoding equine IGF-1 (AdIGF-1) would have a beneficial effect on cartilage healing in an equine femoropatellar joint model. A total of 16 horses underwent arthroscopic repair of a single 15 mm cartilage defect in each femoropatellar joint. One joint received 2 x 10(7) AdIGF-1 modified chondrocytes and the contralateral...
Eizema K, van der Wal DE, van den Burg MM, Dingboom EG, Everts ME.An optimal developed musculoskeletal system is vital for the performance of the horse. Previously, we showed that in the m. gluteus medius from adult untrained horses, identical mRNA and protein expression patterns were found in the majority of fibres. However, co-expression of IIa and IId/x myosin heavy chain (MyHC) was substantially more common at the protein than at the mRNA level, suggesting a transcriptionally controlled fine-tuning of these 2 genes. Objective: To analyse the MyHC transcripts and proteins (including the cardiac alpha isoform) in the same muscle during post natal developme...
Wilke MM, Nydam DV, Nixon AJ.Mesenchymal stem cells (MSCs) provide an important source of pluripotent cells for musculoskeletal tissue repair. This study examined the impact of MSC implantation on cartilage healing characteristics in a large animal model. Twelve full-thickness 15-mm cartilage lesions in the femoropatellar articulations of six young mature horses were repaired by injection of a self-polymerizing autogenous fibrin vehicle containing mesenchymal stem cells, or autogenous fibrin alone in control joints. Arthroscopic second look and defect biopsy was obtained at 30 days, and all animals were euthanized 8 month...
Löhr CV, Juan-Sallés C, Rosas-Rosas A, Parás García A, Garner MM, Teifke JP.Sarcoids were diagnosed in two captive zebras from different facilities. Zebra 1 (Equus burchellii boehmi) was a 4.5-yr-old, captive-born male that presented with a 9- by 7-cm inguinal mass. Seven months after surgical excision of the inguinal mass, the zebra presented with a similar lesion in the right upper eyelid that has relapsed repeatedly and has not responded to treatment including local cisplatin injections and cryosurgery. Zebra 2 (of undetermined taxon) was housed at a private wild animal farm. The zebra presented with a single, raised, 2.5- by 2.0- by 2.0-cm, ulcerated mass on the n...
Sayasith K, Bouchard N, Doré M, Sirois J.The mammalian ovulatory process is accompanied by a gonadotropin-dependent increase in follicular levels of prostaglandin E2 (PGE2) and PGF2alpha, which are metabolized by 15-hydroxy prostaglandin dehydrogenase (PGDH). Little is known about ovarian PGDH regulation in non-primate species. The objectives of this study were to characterize the structure of equine PGDH and its regulation in follicles during human chorionic gonadotropin (hCG)-induced ovulation. The full-length equine PGDH was obtained by RT-PCR, 5'- and 3'-rapid amplification of cDNA ends (RACE). Its open reading frame encodes a 26...
Weston MC, Cunningham FM, Collins ME.CCL11 (also known as eotaxin) is a very potent and selective mediator of eosinophil migration which exerts its effects through its receptor, CCR3. In this study we report the cloning of an equine CCR3 cDNA sequence and investigation of the localization of CCR3 mRNA expression in horse tissues. Equine CCR3 displayed high levels of sequence identity with CCR3 sequences in other species. RT-PCR analysis revealed the expression of CCR3 in colon, lung and spleen of normal horses. In situ hybridisation experiments indicated that expression of CCR3 mRNA in colon was predominantly in eosinophils and t...
Goh G, Raudsepp T, Durkin K, Wagner ML, Schäffer AA, Agarwala R, Tozaki T, Mickelson JR, Chowdhary BP.High-resolution physically ordered gene maps for equine homologs of human chromosome 5 (HSA5), viz., horse chromosomes 14 and 21 (ECA14 and ECA21), were generated by adding 179 new loci (131 gene-specific and 48 microsatellites) to the existing maps of the two chromosomes. The loci were mapped primarily by genotyping on a 5000-rad horse x hamster radiation hybrid panel, of which 28 were mapped by fluorescence in situ hybridization. The approximately fivefold increase in the number of mapped markers on the two chromosomes improves the average resolution of the map to 1 marker/0.9 Mb. The improv...
Borchers K, Ebert M, Fetsch A, Hammond T, Sterner-Kock A.Equine herpes virus 2 (EHV-2), a gamma(2)-herpesvirus, is common in horses of all ages. Its role as a primary pathogen is unclear but there is an association between EHV-2, respiratory disease and keratoconjunctivitis. The purpose of this study was to gain more information on the prevalence of EHV-2 DNA in conjunctival swabs from horses with and without ocular disease and to define the anatomical site and cell type harbouring viral genome or antigen. By polymerase chain reaction (PCR) 22 out of 77 (28.6%) ocular swabs of clinically healthy and only 4 out of 48 (8.3%) samples from diseased hors...
Semevolos SA, Nixon AJ, Fortier LA, Strassheim ML, Haupt J.The purpose of this study was to determine changes in the expression of regulatory molecules in normal equine articular cartilage throughout development up to 18 months of age. The hypothesis was that expression of these regulatory molecules would decrease from birth to postpubescence. Cartilage was harvested from normal femoropatellar or scapulohumeral joints from 34 fresh horse cadavers. Horses were placed in four age groups [prenatal (n = 5); prepubertal, 0-6 months (n = 11); pubertal, 7-14 months (n = 13); and postpubertal, 15-18 months (n = 5)]. Indian hedgehog (Ihh), Gli1, Gli3, Patched1...
Perrocheau M, Boutreux V, Chadi S, Mata X, Decaunes P, Raudsepp T, Durkin K, Incarnato D, Iannuzzi L, Lear TL, Hirota K, Hasegawa T, Zhu B, de Jong P....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...
Swinburne JE, Boursnell M, Hill G, Pettitt L, Allen T, Chowdhary B, Hasegawa T, Kurosawa M, Leeb T, Mashima S, Mickelson JR, Raudsepp T, Tozaki T....A genetic linkage map of the horse consisting of 742 markers, which comprises a single linkage group for each of the autosomes and the X chromosome, is presented. The map has been generated from two three-generation full-sibling reference families, sired by the same stallion, in which there are 61 individuals in the F2 generation. Each linkage group has been assigned to a chromosome and oriented with reference to markers mapped by fluorescence in situ hybridization. The average interval between markers is 3.7 cM and the linkage groups collectively span 2772 cM. The 742 markers comprise 734 mic...
Musilova P, Kubickova S, Vychodilova-Krenkova L, Kralik P, Matiasovic J, Hubertova D, Rubes J, Horin P.Chromosomal locations of 19 horse immunity-related loci (CASP1, CD14, EIF5A, FCER1A, IFNG, IL12A, IL12B, IL12RB2, IL1A, IL23A, IL4, IL6, MMP7, MS4A2, MYD88, NOS2A, PTGS2, TFRC and TLR2) were determined by fluorescence in situ hybridization. For IFNG and PTGS2, this study is a confirmation of their previously reported position. In addition, microsatellite (HMBr1) was localized in the same region as IFNG. All genes were assigned to regions of conserved synteny and the data obtained in this study enhance the comparative human-horse map. Cytogenetic localization of IL6 to ECA4q14-q21.1 suggested a...
Eizema K, van den Burg M, Kiri A, Dingboom EG, van Oudheusden H, Goldspink G, Weijs WA.The horse is one of the few animals kept and bred for its athletic performance and is therefore an interesting model for human sports performance. The regulation of the development of equine locomotion in the first year of life, and the influence of early training on later performance, are largely unknown. The major structural protein in skeletal muscle, myosin heavy-chain (MyHC), is believed to be primarily transcriptionally controlled. To investigate the expression of the MyHC genes at the transcriptional level, we isolated cDNAs encoding the equine MyHC isoforms type 1 (slow), type 2a (fast...
Knight CG, Munday JS, Rosa BV, Kiupel M.A 9-year-old gelding presented with approximately 100 papillomas that covered about 75% of the distal penis. Biopsy was performed, and histology showed evidence of viral cytopathic change and koilocytosis. Polymerase chain reaction using DNA extracted from biopsied tissue amplified equine papillomavirus type 2 (EcPV-2) DNA sequences. Sixteen months later, the horse was re-examined and the appearance of the papillomas was unchanged. Equine papillomavirus type 2 DNA sequences were again amplified from both biopsied tissue and swabs of the penis. Papillomavirus was localized to the lesions by imm...
Smith DJ, Iqbal J, Purewal A, Hamblin AS, Edington N.IL-2 and equine chorionic gonadotrophin (eCG) initiated reactivation of equid herpesvirus-1 (EHV-1) from venous lymphocytes at a frequency of 1/10(-5). Indirect immunofluorescence showed that > 80% of virus-positive leukocytes were CD5+/CD8+ with the remaining 20% being CD5+/CD8-/CD4-. Cocultivation demonstrated that the reactivated virus was infectious. In addition, virus was reactivated in vitro from leukocytes of > 70% of horses by the mitogens phytohaemagglutinin (PHA) and pokeweed mitogen (PWM). Transfer of supernatants showed that IL-2 and eCG acted indirectly by causing the releas...
Löhr CV, Juan-Sallés C, Rosas-Rosas A, Parás García A, Garner MM, Teifke JP.Sarcoids were diagnosed in two captive zebras from different facilities. Zebra 1 (Equus burchellii boehmi) was a 4.5-yr-old, captive-born male that presented with a 9- by 7-cm inguinal mass. Seven months after surgical excision of the inguinal mass, the zebra presented with a similar lesion in the right upper eyelid that has relapsed repeatedly and has not responded to treatment including local cisplatin injections and cryosurgery. Zebra 2 (of undetermined taxon) was housed at a private wild animal farm. The zebra presented with a single, raised, 2.5- by 2.0- by 2.0-cm, ulcerated mass on the n...
de Heus P, Bagó Z, Weidinger P, Lale D, Trachsel DS, Revilla-Fernández S, Matiasek K, Nowotny N.As evidenced by sero-epidemiological studies, infections of horses with the tick-borne encephalitis virus (TBEV) occur frequently in TBEV-endemic areas. However, there are only very few reports of clinical cases. A possible underreporting may be due to a variety of diagnostic challenges. In this study, ELISA and neutralization tests were applied to serum samples. Brain tissue samples were investigated for the presence of nucleic acids of TBEV, Equid alphaherpesvirus 1, Borna disease virus 1, West Nile and Usutu viruses, rustrela virus, as well as Eastern, Western, and Venezuelan equine encepha...
Gerstenberg C, Allen WR, Stewart F.Previous studies showed a dramatic increase in EGF gene expression in the endometrial glands of pregnant mares around day 40 after ovulation. To investigate how the steroid hormones of pregnancy might regulate this expression, in situ hybridization was used to monitor the levels of EGF mRNA in endometrial biopsies obtained from seasonally anoestrous or ovariectomised mares given exogenous progesterone and oestrogen, alone or in combination, for up to 46 days. Biopsies were also taken from mares during the non-pregnant cycle, during normal pregnancies and pregnancies compromised by endometrial ...
Nomura M, Hosaka Y, Kasashima Y, Ueda H, Takehana K, Kuwano A, Arai K.The DNA microarray analysis for matrix metalloproteinase (MMP)-related mRNA expression in equine superficial digital flexor tendinitis indicated that mRNA level of MMP-13 was apparently up-regulated in the tendinitis as compared to normal tendon. In situ hybridization also revealed that fibroblastic cells proliferated in the granulation tissue generated in the tendinitis actively expressed MMP-13 mRNA. On the other hand, in normal tendon, a few fibroblastic cells and vascular components lied in the endotenon barely expressed its mRNA, but other cellular components in the tendon bundle were not...
Greenwood S, Chow-Lockerbie B, Ramsauer S, Wachoski-Dark G, Knight C, Wobeser B. papillomavirus type 2 (EcPV-2) has been recognized as a potential cause of a subset of genital squamous cell carcinomas (SCCs) in horses. In the current study, we measured EcPV-2 seropositivity in 50 healthy horses from Western Canada, and these were compared to a herd of horses with known EcPV-2 exposure. Second, the presence of EcPV-2 DNA was measured using EcPV-2-specific PCR (polymerase chain reaction), performed on a variety of tissues collected at necropsy from 70 horses that lacked any history, gross, or histologic evidence of neoplasia or papillomavirus-associated disease. EcPV-2-spec...
O'Banion MK, Reichmann ME, Sundberg JP.Equine papillomaviruses (EqPV) from naturally occurring cases of cutaneous papillomatosis in several ponies and one horse were isolated, cloned, and characterized. Group specific papillomavirus structural antigens were detected in sections of the papillomas by the peroxidase-antiperoxidase technique, and virions were observed in the in the nuclei of cells in the stratum granulosum and corneum. Negatively stained virions purified from papilloma homogenates by isopycnic CsCl centrifugation were 55 nm in diameter and had typical papillomavirus morphology. The entire viral genomes of two separate ...
Myka JL, Lear TL, Houck ML, Ryder OA, Bailey E.The genus Equus is unusual in that five of the ten extant species have documented centric fission (Robertsonian translocation) polymorphisms within their populations, namely E. hemionus onager, E. hemionus kulan, E. kiang, E. africanus somaliensis, and E. quagga burchelli. Here we report evidence that the polymorphism involves the same homologous chromosome segments in each species, and that these chromosome segments have homology to human chromosome 4 (HSA4). Bacterial artificial chromosome clones containing equine genes SMARCA5 (ECA2q21 homologue to HSA4q31. 21) and UCHL1 (ECA3q22 homologue ...
Pecoraro HL, Felippe MJB, Miller AD, Divers TJ, Simpson KW, Guyer KM, Duhamel GE.Common variable immunodeficiency (CVID) is a rare condition in adult horses characterized by hypogammaglobulinemia and increased susceptibility to parasitic and bacterial infections, including recurrent respiratory diseases, septicemia, and meningitis. Lyme disease is often included as a differential diagnosis in CVID horses with signs of meningitis; however, the Borrelia burgdorferi organism has not been demonstrated previously within central nervous system tissues of CVID horses with neurologic disease, to our knowledge. We report herein a case of neuroborreliosis in a CVID horse, confirmed ...
Sayasith K, Bouchard N, Doré M, Sirois J.The mammalian ovulatory process is accompanied by a gonadotropin-dependent increase in follicular levels of prostaglandin E2 (PGE2) and PGF2alpha, which are metabolized by 15-hydroxy prostaglandin dehydrogenase (PGDH). Little is known about ovarian PGDH regulation in non-primate species. The objectives of this study were to characterize the structure of equine PGDH and its regulation in follicles during human chorionic gonadotropin (hCG)-induced ovulation. The full-length equine PGDH was obtained by RT-PCR, 5'- and 3'-rapid amplification of cDNA ends (RACE). Its open reading frame encodes a 26...
Dahlgren LA, Brower-Toland BD, Nixon AJ.To clone the 5' end of type III collagen and describe its pattern of mRNA and protein expression in normal and healing tendons in horses. Methods: 14 healthy adult horses. Methods: The tensile region of collagenase-injured superficial digital flexor tendons was harvested at intervals from 1 to 24 weeks after injury. Total RNA was reverse-transcribed into cDNA for cloning and sequencing of type III collagen. Equine-specific nucleic acid probes were developed and used for northern blot analysis and in situ hybridization. Type III collagen protein and cyanogen bromide-cleaved collagen peptides we...
van der Haegen A, Griot-Wenk M, Welle M, Busato A, von Tscharner C, Zurbriggen A, Marti E.The aim of the present study was to investigate, with immunohistochemistry and in situ hybridisation, if immunoglobulin-E (IgE) and mast cells are involved in the pathogenesis of insect bite hypersensitivity (IBH), an allergic dermatitis of horses. In tissue sections fixed in paraformaldehyde (PFA) for <24 h, significantly more IgE protein-bearing cells were found in the dermis and epidermis of acute and chronic IBH lesions than in skin biopsies from healthy horses (medians = 466, 236 and 110 cells/mm2, respectively; P < or = 0.01). More IgE-mRNA positive (+) cells were observed in the dermis ...
Ahrens E, Stranzinger G.Previous research revealed that the karyotypes of Equus przewalskii (2n = 66) and Equus caballus (2n = 64) differ by one pair of metacentric chromosomes, present in ECA but not in EPR, and two pairs of acrocentric chromosomes found only in the EPR karyotype. The formation of a trivalent during meiosis in a male F1 hybrid and the homologies in G-banding patterns suggest that ECA 5 corresponds to two acrocentric EPR chromosomes resulting from a Robertsonian fusion or fission event. Chromosomal investigations of a female interspecies F1 hybrid including banded karyograms and fluorescence in situ ...
Lear TL, Lundquist J, Zent WW, Fishback WD, Clark A.Repeated early embryonic loss (REEL) represents a considerable economic loss to the horse industry. Mares that experience REEL may be overlooked as potential carriers of a chromosome abnormality. Here we report three different autosomal translocations in Thoroughbred mares presented for chromosome analysis because of REEL. The karyotypes were 64,XX,t(1;21), 64,XX,t(16;22), and 64,XX,t(4;13), respectively. In order to confirm the chromosomes involved in the translocations, to map the breakpoints, and to determine if the translocations were reciprocal, genes surrounding the breakpoints were iden...
Wijers ER, Zijlstra C, Lenstra JA.The major satellite of the horse genome consists of about 1 million copies of a 221-bp tandem repeat unit. By fluorescence in situ hybridization it has been localized in the centromeres of 58 of the 64 horse chromosomes. The donkey genome contains a similar but not identical satellite. Strikingly, the equine repeat did not hybridize to DNA of the Grevy zebra, despite the divergence of the horse and zebra only 3 to 5 million years ago and the ability of these species to crossbreed. The evolution of satellite DNA in the Equidae is more rapid than that in other mammalian families, which may be ex...
Kambayashi Y, Kishi D, Ueno T, Ohta M, Bannai H, Tsujimura K, Kinoshita Y, Nemoto M.Equine coronavirus (ECoV) causes pyrexia, anorexia, lethargy, and sometimes diarrhoea. Infected horses excrete the virus in their faeces, and ECoV is also detected in nasal samples from febrile horses. However, details about ECoV infection sites in the intestinal and respiratory tracts are lacking. To identify the ECoV infection sites in the intestinal and respiratory tracts, we performed an experimental infection study and analysed intestinal and respiratory samples collected from four infected horses at 3, 5, 7, and 14 days post-inoculation (dpi) by real-time reverse transcription polymeras...
Trenfield K, Spradbrow PB, Vanselow B.DNA was extracted from 14 equine sarcoids, electrophoresed and hybridised with a radioactively labelled probe of bovine papillomavirus type I (BPV 1) DNA under conditions of low stringency. Twelve sarcoids contained sequences of DNA that hybridised with the probe and that comigrated with BPV 2 DNA. The viral DNAs in four of these sarcoids differed from BPV 1 and BPV 2 DNA on restriction endonuclease analysis. One of four cell lines derived from sarcoids also contained BPV 1 related DNA. The results confirm the frequent presence in equine sarcoids of unintegrated papillomaviral DNA and suggest ...
Chowdhary BP, Harbitz I, Davies W, Gustavsson I.In situ hybridization techniques were used to localize regionally the calcium release channel (CRC) gene on cattle and horse chromosomes, using a porcine CRC cDNA probe. In cattle, the hybridization signal peaked on the 18q23-q26 bands and in horse on the 10pter region. Previous studies have shown that the glucose phosphate isomerase (GPI) gene localizes at the same site in both species, indicating that the two loci are syntenic. As CRC and GPI are syntenic in human, pig and mouse, the present results in cattle and horse represent another example of synteny conservation in the evolution of mam...
Hodgkinson JE, Love S, Lichtenfels JR, Palfreman S, Ramsey YH, Matthews JB.Here, we report evaluation of five oligoprobes designed from intergenic spacer (IGS) region sequences for identification of cyathostomin species. Oligoprobes were designed for identification of Cylicocyclus ashworthi, Cylicocyclus nassatus, Cylicostephanus longibursatus, Cylicostephanus goldi and a fifth probe designed to identify all members of this tribe. PCR amplification of IGS DNA from 16 cyathostomin species allowed sequence comparison and identification of four putative species-specific probes. Southern blotting of amplified products from 16 species showed that all probes were species-s...
Raudsepp T, Mariat D, Guérin G, Chowdhary BP.A total of 32 loci comprising specific genes, microsatellites and anonymous BAC clones from horse and cattle were mapped on donkey chromosomes. Of these, 13 markers were also mapped for the first time in the horse. This information, together with that previously available in donkey and horse updates the comparative status of the karyotypes of the two species. The findings of the present study for the first time show correlation between eleven equine acrocentric autosomes and the donkey chromosomes and in part enable detection of rearrangements between them. There are still 7-8 pairs of chromos...
Raudsepp T, Otte K, Rozell B, Chowdhary BP.Three genomic subclones derived from a phage clone containing the equine IGF2 gene were used to FISH map the gene on horse (ECA) and donkey (EAS) metaphase chromosomes. The gene mapped on ECA 12q13 band and is the first locus mapped to this horse chromosome. In donkey the gene mapped very terminal on the long arm of one small submetacentric chromosome that shows almost identical DAPI-banding pattern with ECA12. This is the first locus mapped in donkey genome. Cross species chromosome painting of equine metaphase chromosomes with human Chromosome (Chr) 11-specific probe showed homoeology of thi...
Weissenböck H, Hubálek Z, Halouzka J, Pichlmair A, Maderner A, Fragner K, Kolodziejek J, Loupal G, Kölbl S, Nowotny N.Avian mortality and encephalomyelitis in equines are considered good indicators for West Nile virus (WNV) activity. We retrospectively tested 385 horse sera for WNV antibodies and looked for WNV nucleic acid and/or WNV antigen in paraffin embedded tissues from 12 horses with aetiologically unresolved encephalomyelitis and 102 free-living birds of different species which had been found dead. With the exception of four horses originating from eastern European countries investigated on the occasion of transit through Austria, all horse sera were negative. Nested RT-PCR of the horse tissues yielde...
Klonisch T, Steger K, Kehlen A, Allen WR, Froehlich C, Kauffold J, Bergmann M, Hombach-Klonisch S.We employed molecular and immunological techniques to investigate the expression of INSL3, a member of the insulin-like superfamily, in prepubertal testis, postpubertal testes exhibiting normal and disturbed spermatogenesis, and cryptorchid testes of male horses. In addition, the partial cDNA coding sequences of the equine homologue of the human relaxin/INSL3-receptor Lgr8 were determined. Nonradioactive in-situ hybridization with a cRNA probe for equine Insl3 and immunohistochemistry with a specific rabbit INSL3 antiserum localized Insl3 transcripts and immunoreactive INSL3 ligand to Leydig c...
Maher JK, Tresnan DB, Deacon S, Hannah L, Antczak DF.Down-regulation of major histocompatibility complex (MHC) genes by trophoblast cells is considered to be a primary mechanism preventing maternal immune rejection of the fetal-placental unit in mammalian pregnancy by rendering these cells, which form the primary barrier between mother and fetus, relatively non-antigenic. In situ hybridization with probes encoding human and horse MHC class I genes was used to characterize the pattern of MHC class I mRNA expression in the various forms of horse trophoblast. Strong hybridization signals were observed in the invasive trophoblast cells of chorionic ...
Szczerbal I, Nowacka-Woszuk J, Kopp-Kuhlman C, Mackowski M, Switonski M.X monosomy is the most common disorder of sex development in horses. Although cytogenetic analysis is still the gold standard in the diagnosis of equine X monosomy, novel molecular techniques are being sought to quickly and reliably detect this chromosome abnormality. Objective: The goal of this study was to evaluate the usefulness of a novel variant of the PCR technique-namely, droplet digital PCR (ddPCR)-in the detection of X monosomy in mares. Methods: A proof of concept of the usefulness of ddPCR in diagnosing an abnormal number of X chromosomes in mares. Methods: We examined an infertile ...
Fischer CD, Wachoski-Dark GL, Grant DM, Bramer SA, Klein C.Interferon epsilon (IFNE) is type I interferon which stands out through its unusual expression profile and differing regulation compared to classic type I interferons such as interferon alpha and interferon beta. Unlike other type I interferons, the expression of IFNE is not stimulated through exposure to viral agents. Expression of IFNE is most abundant in mouse and human endometrium where it is constitutively expressed in luminal and glandular epithelial cells and expression levels are up-regulated with estrogen exposure. The aim of the current study was to determine whether a cycle or pregn...
