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Topic:Molecular biology
Molecular biology in horses involves the study of molecular processes and genetic mechanisms that underpin equine physiology and health. This field encompasses the analysis of DNA, RNA, proteins, and other biomolecules to understand gene expression, genetic variation, and cellular functions in horses. Techniques such as genomic sequencing, gene expression profiling, and molecular diagnostics are employed to explore topics like hereditary diseases, performance traits, and immune responses in equines. This page assembles peer-reviewed research studies and scholarly articles that investigate the molecular biology of horses, focusing on genetic research, molecular techniques, and their applications in equine science.
Role of tumor necrosis factor-α, interferon-γ and Fas-ligand on in vitro nitric oxide activity in the corpus luteum. Normal reproductive function involves the expression of inflammatory mediators. Regarding the corpus luteum (CL), cytokines promote the cross-talk between immune, vascular and steroidogenic cells, among others. Moreover, TNF, IFNG and FASL were shown to regulate equine CL establishment and regression. We hypothesized that cytokines action on equine CL may be mediated by nitric oxide (NO), through the regulation of endothelial NO synthase (eNOS) expression. TNF increased eNOS mRNA level and NO metabolite (nitrite) production during CL growth. Cytokines combined action (TNF+IFNG+FASL) promoted e...
Biochemical characterization of stallion prostasomes and comparison to their human counterparts. Release of nanometer-sized prostasomes into human and equine semen suggests essential functions in their relationships with sperm cells and the fertilization process. The two types of prostasomes displayed ultrastructural similarities, albeit the human prostasomes were somewhat larger than the stallion prostasomes. A high ratio of saturated fatty acids was characteristic for the two prostasome types. Electrophoretic separation systems revealed an equine prostasomal pattern different from that of human. The 21 distinctive low molecular weight protein spots in the 2D-gel (with no counterparts in...
Equine oviduct explant culture: a basic model to decipher embryo-maternal communication. Equine embryos remain for 6 days in the oviduct and thus there is a need for an in vitro model to study embryo-oviductal interactions in the horse, since this subtle way of communication is very difficult to analyse in vivo. Until now, no equine oviduct explant culture model has been characterised both morphologically and functionally. Therefore, we established a culture system for equine oviduct explants that maintained epithelial morphology during 6 days of culture, as revealed by light microscopy and transmission electron microscopy. We demonstrated the presence of highly differentiated, ta...
Transcriptome analysis of a parasitic clade V nematode: comparative analysis of potential molecular anthelmintic targets in Cylicostephanus goldi. Clade V nematodes comprise several parasitic species that include the cyathostomins, primary helminth pathogens of horses. Next generation transcriptome datasets are available for eight parasitic clade V nematodes, although no equine parasites are included in this group. Here, we report next generation transcriptome sequencing analysis for the common cyathostomin species, Cylicostephanus goldi. A cDNA library was generated from RNA extracted from 17 C. goldi male and female adult parasites. Following sequencing using a 454 GS FLX pyrosequencer, a total of 475,215 sequencing reads were generate...
Involvement of miRNAs in equine follicle development. Previous evidence from in vitro studies suggests specific roles for a subset of miRNAs, including miR-21, miR-23a, miR-145, miR-503, miR-224, miR-383, miR-378, miR-132, and miR-212, in regulating ovarian follicle development. The objective of this study was to determine changes in the levels of these miRNAs in relation to follicle selection, maturation, and ovulation in the monovular equine ovary. In Experiment 1, follicular fluid was aspirated during ovulatory cycles from the dominant (DO) and largest subordinate (S) follicles of an ovulatory wave and the dominant (DA) follicle of a mid-cycle...
Analysis of unannotated equine transcripts identified by mRNA sequencing. Sequencing of equine mRNA (RNA-seq) identified 428 putative transcripts which do not map to any previously annotated or predicted horse genes. Most of these encode the equine homologs of known protein-coding genes described in other species, yet the potential exists to identify novel and perhaps equine-specific gene structures. A set of 36 transcripts were prioritized for further study by filtering for levels of expression (depth of RNA-seq read coverage), distance from annotated features in the equine genome, the number of putative exons, and patterns of gene expression between tissues. From ...
Differential miRNA expression between equine ovulatory and anovulatory follicles. Relatively little is known about the physiological roles of microRNAs (miRNAs) during follicular development. Previous evidence from in vitro studies suggests specific roles for a subset of miRNAs, including miR-21, miR-23a, miR-145, miR-503, miR-224, miR-383, miR-378, miR-132, and miR-212, in regulating ovarian follicle development. The objective of this study was to gain insight on the involvement of these miRNAs during follicle maturation. Follicular fluid was aspirated from dominant follicles (>32 mm) during the ovulatory season (July to October) and the anovulatory season (January to M...
Design of NK-2-derived peptides with improved activity against equine sarcoid cells. Equine sarcoid is a topically accessible model for the evaluation of anticancer peptides acting by physical membrane disruption avoiding the complexity of a systemic application. We aim at evaluating and improving natural peptides for host defence as lead structures, where we focus on the cationic and amphipathic peptide NK-2. Cytotoxicity tests, fluorescence microscopy and a chip-based biosensor, which enabled real-time monitoring of cell metabolism, were applied. Cancer cell killing was dynamic with an initial phase of increased cellular respiration, followed by membrane destruction. NK-2 wa...
A comparison of three-dimensional culture systems to evaluate in vitro chondrogenesis of equine bone marrow-derived mesenchymal stem cells. To compare in vitro three-dimensional (3D) culture systems that model chondrogenesis of bone marrow-derived mesenchymal stem cells (MSCs). Methods: MSCs from five horses 2-3 years of age were consolidated in fibrin 0.3% alginate, 1.2% alginate, 2.5×10(5) cell pellets, 5×10(5) cell pellets, and 2% agarose, and maintained in chondrogenic medium with supplemental TGF-β1 for 4 weeks. Pellets and media were tested at days 1, 14, and 28 for gene expression of markers of chondrogenic maturation and hypertrophy (ACAN, COL2B, COL10, SOX9, 18S), and evaluated by histology (hematoxylin and eosin, Tolu...
BAG3 protects bovine papillomavirus type 1-transformed equine fibroblasts against pro-death signals. In human cancer cells, BAG3 protein is known to sustain cell survival. Here, for the first time, we demonstrate the expression of BAG3 protein both in equine sarcoids in vivo and in EqS04b cells, a sarcoid-derived fully transformed cell line harbouring bovine papilloma virus (BPV)-1 genome. Evidence of a possible involvement of BAG3 in equine sarcoid carcinogenesis was obtained by immunohistochemistry analysis of tumour samples. We found that most tumour samples stained positive for BAG3, even though to a different grade, while normal dermal fibroblasts from healthy horses displayed very weak ...
The use of bioinspired alterations in the glycosaminoglycan content of collagen-GAG scaffolds to regulate cell activity. The design of biomaterials for regenerative medicine can require biomolecular cues such as growth factors to induce a desired cell activity. Signal molecules are often incorporated into the biomaterial in either freely-diffusible or covalently-bound forms. However, biomolecular environments in vivo are often complex and dynamic. Notably, glycosaminoglycans (GAGs), linear polysaccharides found in the extracellular matrix, are involved in transient sequestration of growth factors via charge interactions. Biomaterials mimicking this phenomenon may offer the potential to amplify local biomolecular...
Recovery of African horse sickness virus from synthetic RNA. African horse sickness virus (AHSV) is an insect-vectored emerging pathogen of equine species. AHSV (nine serotypes) is a member of the genus Orbivirus, with a morphology and coding strategy similar to that of the type member, bluetongue virus. However, these viruses are distinct at the genetic level, in the proteins they encode and in their pathobiology. AHSV infection of horses is highly virulent with a mortality rate of up to 90 %. AHSV is transmitted by Culicoides, a common European insect, and has the potential to emerge in Europe from endemic countries of Africa. As a result, a safe an...
Analysis of activated platelet-derived growth factor β receptor and Ras-MAP kinase pathway in equine sarcoid fibroblasts. Equine sarcoids are skin tumours of fibroblastic origin affecting equids worldwide. Bovine papillomavirus type-1 (BPV-1) and, less commonly, type-2 are recognized as etiological factors of sarcoids. The transforming activity of BPV is related to the functions of its major oncoprotein E5 which binds to the platelet-derived growth factor β receptor (PDGFβR) causing its phosphorylation and activation. In this study, we demonstrate, by coimmunoprecipitation and immunoblotting, that in equine sarcoid derived cell lines PDGFβR is phosphorylated and binds downstream molecules related to Ras-mitoge...
Equine herpesvirus 1 (EHV-1) nucleotide polymorphism determination using formalin fixed tissues in EHV-1 induced abortions and myelopathies with real-time PCR and pyrosequencing. Equine herpesvirus-1 (EHV-1) strains with a single point mutation at the 2254 nucleotide position with a G2254 constitution within the DNA polymerase gene are associated strongly with equine myeloencephalopathies. Infections with non-neuropathogenic EHV-1 strains without the G2254 nucleotide but with an A2254 nucleotide are associated less frequently with equine neurologic disease. A retrospective study utilizing DNA extracted from formalin fixed paraffin embedded tissues was conducted with real time PCR and pyrosequencing, to determine the infecting EHV-1 strains. Infection with EHV-1 A2254 a...
Characterization of glycoproteins in equine herpesvirus-1. In this study, we attempted to express twelve glycoproteins of equine herpesvirus-1 (EHV-1) in 293T cells and to characterize these using monoclonal antibodies (MAbs) and horse sera against EHV-1. Expression of glycoprotein B (gB), gC, gD, gG, gI and gp2 was recognized by immunoblot analysis using horse sera, but that of gE, gH, gK, gL, gM and gN was not. Four MAbs recognized gB, four recognized gC and one recognized gp2. Two MAbs against gB cross-reacted with EHV-4. Interestingly, coexpression of gE and gI and gM and gN enhanced their antigenicity. Furthermore, immunoblot analysis of gp2 show...
Rhodococcus equi: the many facets of a pathogenic actinomycete. Rhodococcus equi is a soil-dwelling pathogenic actinomycete that causes pulmonary and extrapulmonary pyogranulomatous infections in a variety of animal species and people. Young foals are particularly susceptible and develop a life-threatening pneumonic disease that is endemic at many horse-breeding farms worldwide. R. equi is a facultative intracellular parasite of macrophages that replicates within a modified phagocytic vacuole. Its pathogenicity depends on a virulence plasmid that promotes intracellular survival by preventing phagosome-lysosome fusion. Species-specific tropism of R. equi fo...
The molecular basis for recognition of bacterial ligands at equine TLR2, TLR1 and TLR6. TLR2 recognises bacterial lipopeptides and lipoteichoic acid, and forms heterodimers with TLR1 or TLR6. TLR2 is relatively well characterised in mice and humans, with published crystal structures of human TLR2/1/Pam3CSK4 and murine TLR2/6/Pam2CSK4. Equine TLR4 is activated by a different panel of ligands to human and murine TLR4, but less is known about species differences at TLR2. We therefore cloned equine TLR2, TLR1 and TLR6, which showed over 80% sequence identity with these receptors from other mammals, and performed a structure-function analysis. TLR2/1 and TLR2/6 from both horses and hu...
Potential of a sequence-based antigenic distance measure to indicate equine influenza vaccine strain efficacy. The calculation of p(epitope) values, a sequence-based measure of antigenic distance between strains, was developed for human influenza. The potential to apply the p(epitope) value to equine influenza vaccine strain selection was assessed. There was a negative correlation between p(epitope) value and vaccine efficacy for pairs of vaccine and challenge strains used in cross-protection studies in ponies that just reached statistical significance (p=0.046) only if one pair of viruses was excluded from the analysis. Thus the p(epitope) value has potential to provide additional data to consider in ...
Tenascin-C Expression in Equine Tendon-derived Cells During Proliferation and Migration. In vitro cell studies might be a useful tool for studying tendon pathology, but no suitable in vitro models exist for tendon disorders. The purpose of this study was to confirm whether cell scratch culture using tendon-derived fibroblasts can provide a suitable in vitro tendon disorder model. Extracellular matrix components were examined immunohistochemically in tendon tissue, and then their related gene expression levels were analyzed by conventional reverse transcription polymerase chain reaction (RT-PCR) and/or quantitative real-time RT-PCR in tissues and cells. Collagen type I (Col I), col...
Isolation and genetic characterization of naturally NS-truncated H3N8 equine influenza virus in South Korea. Equine influenza virus (EIV) causes a highly contagious respiratory disease in equids, with confirmed outbreaks in Europe, America, North Africa, and Asia. Although China, Mongolia, and Japan have reported equine influenza outbreaks, Korea has not. Since 2011, we have conducted a routine surveillance programme to detect EIV at domestic stud farms, and isolated H3N8 EIV from horses showing respiratory disease symptoms. Here, we characterized the genetic and biological properties of this novel Korean H3N8 EIV isolate. This H3N8 EIV isolate belongs to the Florida sublineage clade 1 of the America...
Transmission of Streptococcus equi subspecies zooepidemicus infection from horses to humans. Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) is a zoonotic pathogen for persons in contact with horses. In horses, S. zooepidemicus is an opportunistic pathogen, but human infections associated with S. zooepidemicus are often severe. Within 6 months in 2011, 3 unrelated cases of severe, disseminated S. zooepidemicus infection occurred in men working with horses in eastern Finland. To clarify the pathogen's epidemiology, we describe the clinical features of the infection in 3 patients and compare the S. zooepidemicus isolates from the human cases with S. zooepidemicus isolates...
Comparative analysis of 16S RNA nucleotide sequences of Anaplasma phagocytophilum detected in the blood of horses from various parts of Europe. The aim of this study was to conduct a comparative analysis of the 16S rRNA gene fragment nucleotide sequences for Anaplasma phagocytophilum strains detected in the blood of horses from various parts of Europe. The study comprised 234 horses that had had contact with ticks. Using PCR, the genetic material of A. phagocytophilum was identified in the blood of 42 animals. The sequences of the 16S RNA gene amplicons that were obtained from our A. phagocytophilum isolates had 100 % similarity with each other and 96.4-100 % similarity with Anaplasma spp. sequences selected from those available i...
The structure of latherin, a surfactant allergen protein from horse sweat and saliva. Latherin is a highly surface-active allergen protein found in the sweat and saliva of horses and other equids. Its surfactant activity is intrinsic to the protein in its native form, and is manifest without associated lipids or glycosylation. Latherin probably functions as a wetting agent in evaporative cooling in horses, but it may also assist in mastication of fibrous food as well as inhibition of microbial biofilms. It is a member of the PLUNC family of proteins abundant in the oral cavity and saliva of mammals, one of which has also been shown to be a surfactant and capable of disrupting m...
Envelope determinants of equine lentiviral vaccine protection. Lentiviral envelope (Env) antigenic variation and associated immune evasion present major obstacles to vaccine development. The concept that Env is a critical determinant for vaccine efficacy is well accepted, however defined correlates of protection associated with Env variation have yet to be determined. We reported an attenuated equine infectious anemia virus (EIAV) vaccine study that directly examined the effect of lentiviral Env sequence variation on vaccine efficacy. The study identified a significant, inverse, linear correlation between vaccine efficacy and increasing divergence of the ...
Reverse mutation of the virulence-associated S2 gene does not cause an attenuated equine infectious anemia virus strain to revert to pathogenicity. The contribution of S2 accessory gene of equine infectious anemia virus (EIAV) to the virulence of pathogenic strains was investigated in the present study by reverse mutation of all four consensus S2 mutation sites in an attenuated EIAV proviral strain, FDDV3-8, to the corresponding sequences of a highly pathogenic strain DV117. The S2 reverse-mutated recombinant strain FDDVS2r1-2-3-4 replicated with similar kinetics to FDDV3-8 in cultivated target cells. In contrast to the results of other studies of EIAV with dysfunctional S2, reverse mutation of S2 only transiently and moderately increased...
Focal adhesion kinases and calcium/calmodulin-dependent protein kinases regulate protein tyrosine phosphorylation in stallion sperm. Protein tyrosine phosphorylation (PY) is a hallmark of sperm capacitation. In stallion sperm, calcium inhibits PY at pH 7.8). These results show for the first time a role for calcium/calmodulin-dependent kinases in PRKA-dependent sperm PY; a non-PRKA-dependent pathway regulating sperm PY; and the apparent involvement of the FAK family of protein tyrosine kinases downstream in both pathways.
Proteomic characterization and cross species comparison of mammalian seminal plasma. Seminal plasma contains a large protein component which has been implicated in the function, transit and survival of spermatozoa within the female reproductive tract. However, the identity of the majority of these proteins remains unknown and a direct comparison between the major domestic mammalian species has yet to be made. As such, the present study characterized and compared the seminal plasma proteomes of cattle, horse, sheep, pig, goat, camel and alpaca. GeLC-MS/MS and shotgun proteomic analysis by 2D-LC-MS/MS identified a total of 302 proteins in the seminal plasma of the chosen mammali...
Characterization and protective immunogenicity of the SzM protein of Streptococcus zooepidemicus NC78 from a clonal outbreak of equine respiratory disease. Streptococcus zooepidemicus of Lancefield group C is a highly variable tonsillar and mucosal commensal that usually is associated with opportunistic infections of the respiratory tract of vertebrate hosts. More-virulent clones have caused epizootics of severe respiratory disease in dogs and horses. The virulence factors of these strains are poorly understood. The antiphagocytic protein SeM is a major virulence factor and protective antigen of Streptococcus equi, a clonal biovar of an ancestral S. zooepidemicus strain. Although the genome of S. zooepidemicus strain H70, an equine isolate, conta...
Lineages of Streptococcus equi ssp. equi in the Irish equine industry. Streptococcus equi ssp. equi is the causative agent of 'Strangles' in horses. This is a debilitating condition leading to economic loss, yard closures and cancellation of equestrian events. There are multiple genotypes of S. equi ssp. equi which can cause disease, but to date there has been no systematic study of strains which are prevalent in Ireland. This study identified and classified Streptococcus equi ssp. equi strains isolated from within the Irish equine industry. Results: Two hundred veterinary isolates were subjected to SLST (single locus sequence typing) based on an internal sequenc...
A new genomic tool, ultra-frequently cleaving TaqII/sinefungin endonuclease with a combined 2.9-bp recognition site, applied to the construction of horse DNA libraries. Genomics and metagenomics are currently leading research areas, with DNA sequences accumulating at an exponential rate. Although enormous advances in DNA sequencing technologies are taking place, progress is frequently limited by factors such as genomic contig assembly and generation of representative libraries. A number of DNA fragmentation methods, such as hydrodynamic sharing, sonication or DNase I fragmentation, have various drawbacks, including DNA damage, poor fragmentation control, irreproducibility and non-overlapping DNA segment representation. Improvements in these limited DNA scissi...
