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Topic:In Situ Hybridization
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.
Molecular Cytogenetic and Y Copy Number Analysis of a Reciprocal ECAY-ECA13 Translocation in a Stallion with Complete Meiotic Arrest. We present a detailed molecular cytogenetic analysis of a reciprocal translocation between horse (ECA) chromosomes Y and 13 in a Friesian stallion with complete meiotic arrest and azoospermia. We use dual-color fluorescence in situ hybridization with select ECAY and ECA13 markers and show that the translocation breakpoint in ECAY is in the multicopy region and in ECA13, at the centromere. One resulting derivative chromosome, Y;13p, comprises of ECAY heterochromatin (ETSTY7 array), a small single copy and partial Y multicopy region, and ECA13p. Another derivative chromosome 13q;Y comprises of E...
Transcriptional profiling of equine endometrium before, during and after capsule disintegration during normal pregnancy and after oxytocin-induced luteostasis in non-pregnant mares. The current study used RNA sequencing to determine transcriptional profiles of equine endometrium collected 14, 22, and 28 days after ovulation from pregnant mares. In addition, the transcriptomes of endometrial samples obtained 20 days after ovulation from pregnant mares, and from non-pregnant mares which displayed and failed to display extended luteal function following the administration of oxytocin, were determined and compared in order to delineate genes whose expressions depend on the presence of the conceptus as opposed to elevated progesterone alone. A mere fifty-five transcripts were ...
First Clinical Case of Equine Parvovirus-Hepatitis-Related Theiler’s Disease in Asia. Equine parvovirus-hepatitis (EqPV-H) is a newly identified etiologic agent of Theiler's disease (TD). We present a case of EqPV-H-related fulminant hepatitis in a 14-year-old thoroughbred mare in Korea. The mare had acute hepatopathy and gastrointestinal symptoms, with abnormal liver-related blood parameters. The horse was born in the USA and imported to Korea in 2017, with no history of administration of equine biological products after entry into Korea. The horse was diagnosed with EqPV-H-associated hepatitis after abdominal ultrasonography, laparotomy, and nested polymerase chain reaction (...
Interleukin-6 and lactate dehydrogenase expression in a novel ex vivo rocking model of equine corneal epithelial wound healing. To establish a physiologically relevant ex vivo model of equine corneal epithelial wound healing. Methods: Fourteen equine corneas were randomly assigned to one of two groups: wounded (n = 8) or unwounded (n = 6) controls. In the wounded group, the axial corneal epithelium was removed by applying a 6 mm filter paper disk soaked in 1N-NaOH for 60 s. Corneas were subsequently cultured using an air-liquid interface model. Evaluation of corneal healing was performed daily, and culture medium was collected. Corneas were randomly assigned to undergo processing via histopathology and RNAscope i...
Vulvar squamous cell carcinoma associated with Equus caballus papillomavirus type 2 infection in a Japanese mare. Equus caballus papillomavirus type 2 (EcPV2) infection has been associated with genital squamous cell carcinoma (SCC) development in horses. However, very few reports on EcPV2-associated disease in Asia exist. Our study characterizes pathological and virological features of an EcPV2-associated vulvar SCC from a Japanese mare. Conventional PCR, in situ hybridization, reverse-transcriptase PCR and immunohistochemistry confirmed the presence and distribution of EcPV2 within the lesion and suggested that p53 degradation may not be the mechanism by which this virus induces neoplastic transformation...
Tick-Borne Encephalitis Virus (TBEV) Infection in Two Horses. A final diagnosis in a horse with clinical signs of encephalopathy can be challenging despite the use of extensive diagnostics. Clinical signs are often not pathognomonic and need to be interpreted in combination with (specific) laboratory results and epidemiological data of the geographical region of the origin of the case(s). Here we describe the diagnostic pathway of tick-borne encephalitis virus infection in two horses using established molecular diagnostic methods and a novel in situ hybridization technique to differentiate between regionally important/emerging diseases for central Europe...
Differential Expression Pattern of Retroviral Envelope Gene in the Equine Placenta. Endogenous retroviruses (ERVs) are proviral phases of exogenous retroviruses, which have coevolved with vertebrate genomes for millions of years. The conservation of ERV genes throughout evolution suggests their beneficial effects on their hosts' survival. An example of such positive selection is demonstrated by the syncytin gene, which encodes a protein with affinity for various mammalian placentas that is involved in the formation of syncytiotrophoblasts. Although the horse has an epitheliochorial placenta, in which the fetal trophoblasts are simply apposed to the intact uterine epithelium, ...
Fibroblast-associated protein-α expression and BPV nucleic acid distribution in equine sarcoids. Sarcoids are the most common cutaneous tumor of equids and are caused by bovine papillomavirus (BPV). Different clinical subtypes of sarcoids are well characterized clinically but not histologically, and it is not known whether viral activity influences the clinical or histological appearance of the tumors. The aim of this study was to verify whether the development of different clinical types of sarcoids or the presence of certain histological features were associated with BPV distribution within the tumor. The presence of BPV was assessed by polymerase chain reaction (PCR) and visualized in ...
Prevalence of Equus caballus Papillomavirus Type-2 Infection and Seropositivity in Asymptomatic Western Canadian Horses. 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...
Prevalence and Prognostic Impact of Equus caballus Papillomavirus Type 2 Infection in Equine Squamous Cell Carcinomas in Western Canadian Horses. Equus caballus papillomavirus type-2 (EcPV-2) has been proposed as a causal factor in equine genital squamous cell carcinoma (SCC). This study had 2 objectives: first, calculate the frequency of papillomavirus (PV) and EcPV-2 infection in papillomas, carcinomas in situ (CIS), and SCCs in Western Canadian horses; and second, determine if EcPV-2 status of equine SCCs is associated with overall survival (OS). EcPV-2 status of 115 archived tissue samples, spanning 6 years, was determined using broad spectrum (MY09/11) and EcPV-2-specific polymerase chain reaction (PCR) assays, EcPV-2-E6/E7 chromog...
First report of equine parvovirus-hepatitis-associated Theiler’s disease in Europe. Equine parvovirus-hepatitis (EqPV-H) has been proposed as the aetiological cause of Theiler's disease, also known as serum hepatitis. EqPV-H-associated Theiler's disease has not been previously reported in Europe. Objective: To determine whether EqPV-H infection was associated with a 2018-2019 outbreak of Theiler's disease in four horses on a studfarm. Methods: Descriptive case series. Methods: The medical records of four horses from the same farm diagnosed with fatal Theiler's disease were examined retrospectively. Information collected included a clinical history, physical examination findin...
A Subset of Equine Gastric Squamous Cell Carcinomas Is Associated With Equus Caballus Papillomavirus-2 Infection. Squamous cell carcinoma (SCC) is the most common neoplasm of the equine stomach. However, the mechanisms underlying malignant transformation are unknown. As papillomavirus-2 (EcPV-2) is a likely cause of some genital SCCs, we hypothesized that EcPV-2 is associated with a subset of equine gastric SCCs. To this aim, we performed polymerase chain reaction (PCR) and in situ hybridization (ISH) for EcPV-2 E6/ E7 oncogenes on 11 gastric SCCs and on gastric samples from 15 control horses with no SCC. PCR for EcPV-2 was positive in 7/11 (64%) gastric SCCs; non-SCC gastric samples were all negative. I...
Application of droplet digital PCR in diagnosing of X monosomy in mares. 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 ...
Premature centromere division (PCD) identified in a hucul mare with reproductive difficulties. A hucul mare with reproductive abnormalities was examined during karyotype analysis. The karyotype was analysed based on evaluation of 860 metaphase plates in chromosome preparations. The use of fluorescence in situ hybridization (FISH) with an X chromosome painting probe showed premature X chromosome separation in 9.5% cases of examined chromosome spreads. In this report, we present the first identify premature centromere division (PCD) as a possible cause of abnormal X chromosome morphology in horses and as a probable cause of reproductive difficulties.
Expression Profile of the Chromosome 14 MicroRNA Cluster (C14MC) Ortholog in Equine Maternal Circulation throughout Pregnancy and Its Potential Implications. Equine chromosome 24 microRNA cluster (C24MC), the ortholog of human C14MC, is a pregnancy-related miRNA cluster. This cluster is believed to be implicated in embryonic, fetal, and placental development. The current study aimed to characterize the expression profile of this cluster in maternal circulation throughout equine gestation. The expression profile of miRNAs belonging to this cluster was analyzed in the serum of non-pregnant (diestrus), pregnant (25 d, 45 d, 4 mo, 6 mo, 10 mo), and postpartum mares. Among the miRNAs examined, 11 miRNAs were differentially expressed across the analyzed ...
Paving the way for more precise diagnosis of EcPV2-associated equine penile lesions. There is growing evidence that equine papillomavirus type 2 (EcPV2) infection is causally associated with the development of equine genital squamous cell carcinomas (SCCs). Early stages of disease present clinically as plaques or wart-like lesions which can gradually progress to tumoural lesions. Histologically these lesions are inconsistently described as benign hyperplasia, papilloma, penile intraepithelial neoplasia (PIN), carcinoma in situ (CIS) or SCC. Guidelines for histological classification of early SCC precursor lesions are not precisely defined, leading to potential misdiagnosis. Th...
The expression of equine keratins K42 and K124 is restricted to the hoof epidermal lamellae of Equus caballus. The equine hoof inner epithelium is folded into primary and secondary epidermal lamellae which increase the dermo-epidermal junction surface area of the hoof and can be affected by laminitis, a common disease of equids. Two keratin proteins (K), K42 and K124, are the most abundant keratins in the hoof lamellar tissue of Equus caballus. We hypothesize that these keratins are lamellar tissue-specific and could serve as differentiation- and disease-specific markers. Our objective was to characterize the expression of K42 and K124 in equine stratified epithelia and to generate monoclonal antibodie...
Azoospermia and Y Chromosome-Autosome Translocation in a Friesian Stallion. This case report describes spermatogenic arrest and azoospermia in a stallion with a unique Y chromosome-autosome translocation. Clinical diagnosis of azoospermia was based on history of infertility and evaluation of ejaculates collected for artificial insemination. Clinical and ultrasonographic evaluation of the external and internal genitalia did not reveal any abnormalities except for smaller than normal testicular size. Azoospermia of testicular origin was confirmed by determining alkaline phosphatase concentration in semen. Histological evaluation of testicular tissue after castration con...
Chronic equine hepacivirus infection in an adult gelding with severe hepatopathy. Equine hepacivirus (EqHV) in equids represents the closest homologue to hepatitis C virus (HCV) infecting humans. A majority of HCV infected patients develop a chronic course of infection leading to liver fibrosis, cirrhosis and liver failure. However, in horses mostly transient mild subclinical infections are reported for EqHV to date. EqHV can be involved in chronic liver diseases of horses. Biochemical parameters in serum samples were measured. Viral load was determined using qPCR. Next generation sequencing (NGS) of serum was performed. Liver tissue was stained with haematoxylin and eosin ...
Satellite DNA at the Centromere is Dispensable for Segregation Fidelity. The typical vertebrate centromeres contain long stretches of highly repeated DNA sequences (satellite DNA). We previously demonstrated that the karyotypes of the species belonging to the genus are characterized by the presence of satellite-free and satellite-based centromeres and represent a unique biological model for the study of centromere organization and behavior. Using horse primary fibroblasts cultured in vitro, we compared the segregation fidelity of chromosome 11, whose centromere is satellite-free, with that of chromosome 13, which has similar size and a centromere containing long s...
Fertility and 63,X Mosaicism in a Haflinger Sibship. Chromosomal abnormalities are notable causes of infertility in horses. Mares show various degrees of estrous behavior, and ultrasound examination often reveals an underdeveloped genital tract. This article reports investigations on fertility in a Haflinger sibship with a healthy, normally developed, fertile mare with at least three healthy offspring. Chromosomal analysis performed incidentally and blinded for this mare revealed 63,X/64,XX/65,XXX mosaicism. Two closely related mares were also mosaics (63,X/64,XX), and one of them was a carrier of a marker chromosome. Repeated examinations of th...
Lawsonia intracellularis associated equine proliferative enteropathy in Danish weanling foals. Lawsonia intracellularis, an obligate intracellular bacterium, causes equine proliferative enteropathy, mainly in horses around weaning. This disease is rarely reported in the Scandinavian countries. Results: Five cases of equine proliferative enteropathy were diagnosed between 2008-2016 at the University of Copenhagen Large Animal Teaching Hospital. Cases were Danish Warmbloods and a Friesian horse, aged 6-7 months, presenting with typical clinical signs of lethargy, poor body condition, pyrexia and diarrhea. Clinical pathology was consistent with previous reports of severe hypoalbuminemia a...
Neuroborreliosis in a horse with common variable immunodeficiency. 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 ...
Kinetics of the chromosome 14 microRNA cluster ortholog and its potential role during placental development in the pregnant mare. The human chromosome 14 microRNA cluster (C14MC) is a conserved microRNA (miRNA) cluster across eutherian mammals, reported to play an important role in placental development. However, the expression kinetics and function of this cluster in the mammalian placenta are poorly understood. Here, we evaluated the expression kinetics of the equine C24MC, ortholog to the human C14MC, in the chorioallantoic membrane during the course of gestation. Results: We demonstrated that C24MC-associated miRNAs presented a higher expression level during early stages of pregnancy, followed by a decline later in g...
Interferon epsilon is constitutively expressed in equine endometrium and up-regulated during the luteal phase. 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...
Birth, evolution, and transmission of satellite-free mammalian centromeric domains. Mammalian centromeres are associated with highly repetitive DNA (satellite DNA), which has so far hindered molecular analysis of this chromatin domain. Centromeres are epigenetically specified, and binding of the CENPA protein is their main determinant. In previous work, we described the first example of a natural satellite-free centromere on Chromosome 11. Here, we investigated the satellite-free centromeres of by using ChIP-seq with anti-CENPA antibodies. We identified an extraordinarily high number of centromeres lacking satellite DNA (16 of 31). All of them lay in LINE- and AT-rich regio...
Milk-fat globule epidermal growth factor 8 (MFGE8) is expressed at the embryo- and fetal-maternal interface in equine pregnancy. Milk-fat globule epidermal growth factor (EGF) 8 protein (MFGE8), also known as lactadherin, promotes cell adhesion in an Arg-Gly-Asp (RGD)-dependent modus via integrins. In the present study, the expression of MFGE8 was examined in equine endometrium during oestrus and at Days 12 and 16 after ovulation in pregnant and non-pregnant mares and in mares during the 5th month of gestation. Results demonstrated that MFGE8 is expressed at the embryo- and fetal-maternal interface in equine pregnancy. In non-pregnant endometrium its expression was upregulated by oestrogen, a finding that was confirmed ...
The occurrence of biofilm in an equine experimental wound model of healing by secondary intention. In humans, biofilm is a well-known cause of delayed healing and low-grade inflammation of chronic wounds. In horses, biofilm formation in wounds has been studied to a very limited degree. The objective of this study was thus to investigate the occurrence of biofilm in equine experimental wounds healing by secondary intention. Tissue biopsies from non-contaminated, experimental excisional shoulder and limb wounds were obtained on day 1-2, day 7-10 and day 14-15 post-wounding. Limb wounds were either un-bandaged or bandaged to induce exuberant granulation tissue (EGT) formation and thereby impai...
First case of sterility associated with sex chromosomal abnormalities in a jenny. Chromosomal abnormalities are one of the main causes of genetic infertility in horses. Currently, their detection rate is rising due to the use of new diagnostic tools employing molecular markers linked to the sex chromosome pair. Despite genetic similarities, there are no previous reports of sterility associated with chromosomal abnormalities in the domestic donkey (Equus asinus). Hereby, we determined the presence of a chromosomal mosaicism in a female donkey with reproductive problems using molecular methodologies developed for horses. A two-and-a-half-year-old jenny characterized by morpho...
Detection of equine arteritis virus by two chromogenic RNA in situ hybridization assays (conventional and RNAscope(®)) and assessment of their performance in tissues from aborted equine fetuses. Equine arteritis virus (EAV) is the causative agent of equine viral arteritis, a respiratory and reproductive disease of equids. EAV infection can induce abortion in pregnant mares, fulminant bronchointerstitial pneumonia in foals, and persistent infection in stallions. Here, we developed two RNA in situ hybridization (ISH) assays (conventional and RNAscope(®) ISH) for the detection of viral RNA in formalin-fixed paraffin-embedded (FFPE) tissues and evaluated and compared their performance with nucleocapsid-specific immunohistochemistry (IHC) and virus isolation (VI; gold standard) techniques...
