Western blotting is an analytical technique used to detect specific proteins in a sample through the use of gel electrophoresis and antibody-based detection. In equine research, this method is employed to study protein expression, post-translational modifications, and protein-protein interactions in horses. The technique involves the separation of proteins by size using polyacrylamide gel electrophoresis, followed by their transfer to a membrane, and subsequent identification using specific antibodies. Western blotting is utilized in various studies to investigate equine diseases, immune responses, and muscle physiology. This page compiles peer-reviewed research studies and scholarly articles that explore the application, methodology, and findings of Western blot analyses in equine science.
Liu ZQ, Xia J, Wang GL, Kuermanali N.Hyalomma asiaticum tick, an important ectozoic parasite causes tickle, pain, anemia, weight loss, and paralysis in its hosts, which include humans, cattle, sheep, horses, camels, and hares. The 4D8 gene can be a potential vaccine candidate antigen for H. asiaticum. In the present study, we cloned and expressed the 4D8 gene of H. asiaticum from Xinjiang Province. Primers were designed according to the H. asiaticum tick 4D8 gene sequence available in GenBank. The gene was amplified by reverse transcription-polymerase chain reaction and the fragments were subcloned into the prokaryotic expression...
Takesue M, Osaka Y, Muranaka M, Katayama Y, Ikadai H.In the present study, the serum and cerebrospinal fluid of horses diagnosed with Setaria digitata cerebrospinal filariasis were analyzed by western blot. The results revealed S. digitata protein bands measuring 65, 34, 22, and 18 kDa in molecular weight. In particular, the 18 kDa band is a possible candidate for clinical immunodiagnosis on the basis of western blot findings.
Mort JS, Beaudry F, Théroux K, Emmott AA, Richard H, Fisher WD, Lee ER, Poole AR, Laverty S.To characterize the initial events in the cleavage of type II collagen mediated by cathepsin K and demonstrate the presence of the resulting products in human and equine articular osteoarthritic cartilage. Equine type II collagen was digested with cathepsin K and the cleavage products characterized by mass spectrometry. Anti-neoepitope antibodies were raised against the most N-terminal cleavage products and used to investigate the progress of collagen cleavage, in vitro, and the presence of cathepsin K-derived products in equine and human osteoarthritic cartilage. Six cathepsin K cleavage sit...
Lee B, Kang HY, Lee DO, Ahn C, Jeung EB.Claudins, which are known as transmembrane proteins play an essential role in tight junctions (TJs) to form physical barriers and regulate paracellular transportation. To understand equine diseases, it is helpful to measure the tissue-specific expression of TJs in horses. Major equine diseases such as colic and West Nile cause damage to TJs. In this study, the expression level and distribution of claudin-1, -2, -4, and -5 in eight tissues were assessed by Western blotting and immunohistochemistry methods. Claudin-1 was primarily identified in the lung, duodenum, and uterus, claudin-2 was evenl...
Aparicio IM, Martin Muñoz P, Salido GM, Peña FJ, Tapia JA.Use of cooled and frozen semen is becoming increasingly prevalent in the equine industry. However, these procedures cause harmful effects in the sperm cell resulting in reduced cell lifespan and fertility rates. Apoptosis and necrosis-related events are increased during semen cryopreservation. However, a third type of cell death, named autophagy, has not been studied during equine semen storage. Light chain (LC)3 protein is a key component of the autophagy pathway. Under autophagy activation, LC3-I is lipidated and converted to LC3-II. The ratio of LC3-II/LC3-I is widely used as a marker of au...
McGorum BC, Scholes S, Milne EM, Eaton SL, Wishart TM, Poxton IR, Moss S, Wernery U, Davey T, Harris JB, Pirie RS.Equine grass sickness (EGS) is of unknown aetiology. Despite some evidence suggesting that it represents a toxico-infection with Clostridium botulinum types C and/or D, the effect of EGS on the functional targets of botulinum neurotoxins, namely the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, is unknown. Further, while it is commonly stated that, unlike EGS, equine botulism is not associated with autonomic and enteric neurodegeneration, this has not been definitively assessed. Objective: To determine: 1) whether botulism causes autonomic and enteric neurodeg...
Jacobsen S, Ladefoged S, Berg LC.To investigate the role of the major equine acute phase protein serum amyloid A (SAA) in inflammation of equine intraarticular tissues. Methods: Articular chondrocytes and fibroblast-like synoviocytes (FLSs) from 8 horses (4 horses/cell type). Methods: Chondrocytes and FLSs were stimulated in vitro for various periods up to 48 hours with cytokines (recombinant interleukin [IL]-1β, IL-6, tumor necrosis factor-α, or a combination of all 3 [IIT]) or with recombinant SAA. Gene expression of SAA, IL-6, matrix metalloproteinases (MMP)-1 and -3, and cartilage-derived retinoic acid-sensitive protein...
Svala E, Jin C, Rüetschi U, Ekman S, Lindahl A, Karlsson NG, Skiöldebrand E.The glycoprotein lubricin contributes to the boundary lubrication of the articular cartilage surface. The early events of osteoarthritis involve the superficial layer where lubricin is synthesised. Objective: To characterise the glycosylation profile of lubricin in synovial fluid from horses with osteoarthritis and study secretion and degradation of lubricin in an in vitro inflammation cartilage model. Methods: In vitro study. Methods: Synovial fluid samples collected from horses with joints with normal articular cartilage and structural osteoarthritic lesions; with and without osteochondral f...
Kim JT, Jung HJ, Song H, Yoon MJ.Acrosin Binding Protein (ACRBP) is specifically localized in the acrosome of germ cells of several species, including mice, pigs, guinea pigs, and humans. The main objective of this study was to investigate ACRBP patterns in the germ cells of stallions at different reproductive stages and seminiferous tubule stages using Western blot, immunohistochemistry, and immunocytochemistry techniques. The stallion reproductive stages were classified as follows: pre-pubertal and post-pubertal stages based on the presence/absence of lumen opening in the seminiferous tubules and full spermatogenesis. The p...
Kim JY, Jung HJ, Yoon MJ.Expression of the protein DDX4/MVH, or VASA, has been reported in germ cells of several species. The main objectives of this study were to (i) investigate VASA expression patterns in testicular cells of stallions at two different reproductive stages (pre-pubertal and post-pubertal) and (ii) evaluate the use of VASA antibody as a molecular marker for single germ cells from stallions. Testicular tissues were obtained from stallions and categorized as pre-pubertal and post-pubertal based on the formation of lumen and status of spermatogenesis on the cross section of seminiferous tubules. The resu...
Chung CJ, Grimm AL, Wilson CL, Balasuriya UB, Chung G, Timoney PJ, Bandaranayaka-Mudiyanselage CB, Lee SS, McGuire TC.In an effort to improve a competitive blocking enzyme-linked immunosorbent assay (cELISA) for antibody detection to Equine arteritis virus (EAV), antigen purified by anion-exchange membrane chromatography capsule (AEC) was evaluated. Virus purification by the AEC method was rapid and easily scalable. A comparison was made between virus purified by the AEC method with that obtained by differential centrifugation based on the following: 1) the relative purity and quality of EAV glycoprotein 5 (GP5) containing the epitope defined by monoclonal antibody 17B7, and 2) the relative sensitivity of a c...
McGorum BC, Pirie RS, Eaton SL, Keen JA, Cumyn EM, Arnott DM, Chen W, Lamont DJ, Graham LC, Llavero Hurtado M, Pemberton A, Wishart TM.Equine grass sickness (EGS) is an acute, predominantly fatal, multiple system neuropathy of grazing horses with reported incidence rates of ∼2%. An apparently identical disease occurs in multiple species, including but not limited to cats, dogs, and rabbits. Although the precise etiology remains unclear, ultrastructural findings have suggested that the primary lesion lies in the glycoprotein biosynthetic pathway of specific neuronal populations. The goal of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS. Here, we use a bottom-up approach be...
Chen J, Guo X, Li L.The nucleocapsid (N) protein is the most conserved structural protein in equine arteritis virus (EAV). This study aimed to identify the minimal conserved B cell epitope on the EAV N protein. The purified N protein was used to immunize mice for preparing monoclonal antibody (mAb). The reactivity of mAb was evaluated by Western blot and immunofluorescence assay. Moreover, 11 overlapping peptides (named MBP-N1 to MBP-N11) were designed to localize the linear antigenic epitope within the N protein. The peptides were identified by indirect enzyme-linked immunosorbent assay (ELISA) and Western blot....
Miskovic Feutz M, Couetil LL, Riley CP, Zhang X, Adamec J, Raskin RE.Lower expression of secretoglobin and transferrin has been found in the bronchoalveolar lavage fluid (BALF) of a small number of horses with experimentally induced signs of recurrent airway obstruction (RAO) compared to healthy controls. Objective: Secretoglobin and transferrin BALF expression will be similarly decreased in horses with naturally occurring clinical signs of RAO and in horses with experimentally induced clinical signs of RAO as compared to healthy controls and intermediate in horses with inflammatory airway disease (IAD). Methods: Recurrent airway obstruction-affected and contro...
Rapacz-Leonard A, Kankofer M, Leonard M, Wawrzykowski J, Dąbrowska M, Raś A, Paździor-Czapula K, Janowski T.In mammals, placenta separation at term may involve degradation of the extracellular matrix by matrix metalloproteinases (MMPs). The activity of MMPs is modulated by TIMPs. We hypothesized that the placentas of mares that deliver fetal membranes physiologically and those that retain fetal membranes (FMR) differ in terms of histology; mRNA expression of MMP-2 and MMP-9; protein expression of MMP-2, MMP-9, and TIMP-2; and the potential activity of both MMPs. Methods: Placenta biopsies were taken from mares (n = 9; 4 FMR, 5 controls) immediately after foal expulsion. Retention was defined as fail...
Elyasi Gorji Z, Amiri-Yekta A, Gourabi H, Hassani S, Fatemi N, Zerehdaran S, Vakhshiteh F, Sanati MH.Follicle stimulating hormone (FSH) plays an essential role in reproductive physiology and follicular development. Objective: A new variant of the equine () gene was cloned, sequenced, and expressed in ) GS115 yeast expression system. Methods: The full-length cDNAs of the and chains were amplified by reverse transcription polymerase chain reaction (RT-PCR) using the total RNA isolated from an Iranian Turkmen-thoroughbred horse's anterior pituitary gland. The amplified chains were cloned into the pPIC9 vector and transferred into The secretion of recombined eFSH using expression system was...
Rode K, Sieme H, Richterich P, Brehm R.The formation of the blood-testis barrier (BTB) is defined as occurring with the first appearance of spermatocytes at around puberty and is vital for normal spermatogenesis. This barrier between two adjacent Sertoli cells (SCs) consists of a cell junctional protein complex, which includes tight junctions (TJs), adherens junctions, and gap junctions. In many mammalian species, BTB composition has already been investigated, whereas little is known about the equine BTB. In the present study, immunohistochemistry and qualitative Western Blot analysis were used to assess the expression and distribu...
Linardi RL, Megee SO, Mainardi SR, Senoo M, Galantino-Homer HL.The limited characterization of equine skin, eye and hoof epithelial stem cell (ESC) and differentiation markers impedes the investigation of the physiology and pathophysiology of these tissues. Objective: To characterize ESC and differentiation marker expression in epithelial tissues of the equine eye, haired skin and hoof capsule. Methods: Indirect immunofluorescence microscopy and immunoblotting were used to detect expression and tissue localization of keratin (K) isoforms K3, K10, K14 and K124, the transcription factor p63 (a marker of ESCs) and phosphorylated p63 [pp63; a marker of ESC tr...
Rapacz-Leonard A, Raś A, Całka J, Janowski TE.Fetal membrane retention can be a life-threatening condition and its incidence exceeds 50% in heavy draught mares. Although fetal membrane retention is commonly treated with repeated injections of oxytocin, based on the suggestion that it is caused mainly by secondary atony of the uterus, this treatment sometimes fails. This led us to ask if expression of oxytocin receptors differs in mares that retain fetal membranes due to secondary uterine atony. Objective: To determine whether expression of oxytocin receptors in equine placental tissues differs when heavy draught mares expel fetal membrane...
Peffers MJ, McDermott B, Clegg PD, Riggs CM.The aim of the study was to characterise the protein complement of synovial fluid (SF) in health and osteoarthritis (OA) using liquid chromatography mass spectrometry (LC-MS/MS) following peptide-based depletion of high abundance proteins. Methods: SF was used from nine normal and nine OA Thoroughbred horses. Samples were analysed with LC-MS/MS using a NanoAcquity™ LC coupled to an LTQ Orbitrap Velos. In order to enrich the lower-abundance protein fractions protein equalisation was first undertaken using ProteoMiner™. Progenesis-QI™ LC-MS software was used for label-free quantification. ...
Scarlet D, Walter I, Hlavaty J, Aurich C.In mares, FSH and its receptor (FSHR) are essential for ovarian function. The objective of the present study was to analyse FSHR gene expression at the mRNA and protein levels in ovarian tissue from newborn and adult horses. Expression of mRNA was analysed by reverse transcription polymerase chain reaction, whereas FSHR protein was visualised by immunohistochemistry (IHC), immunofluorescence labelling (IF) and western blot. FSHR mRNA was detected in ovarian follicles and luteal tissue from adult mares, as well as in the ovaries of neonates. Follicular growth up to 4mm in diameter was already p...
Espino-Solis GP, Quintero-Hernandez V, Olvera-Rodriguez A, Calderon-Amador J, Pedraza-Escalona M, Licea-Navarro A, Flores-Romo L, Possani LD.The αX I-domain of the horse integrin CD11c was successfully expressed in Escherichia coli, purified, biochemically characterized and used as immunogen to generate murine monoclonal antibodies against horse CD11c, which are not yet commercially available. One monoclonal antibody mAb-1C4 against the αX I-domain, is an IgG2a able to interact with the recombinant I-domain, showing an EC50=2.4ng according to ELISA assays. By western blot with horse PBMCs lysates the mAb-1C4 recognized a protein of 150kDa which corresponds well with the CD11c molecule. Using immunohistochemistry in horse lymph no...
Caporali EH, Kuykendall T, Stewart MC.To fully sequence and characterize equine aggrecan and confirm conservation of major aggrecanase, calpain and matrix metalloproteinase (MMP) cleavage sites. Methods: Reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends were used to generate clones that encompassed the complete equine aggrecan sequence. Clones were sequenced and compared with the equine genome database to determine intron-exon boundaries. Results: The aggrecan gene spans over 61 kb on chromosome 1 and is encoded by 17 exons. Two major variants of aggrecan were cloned; one containing 8187 bp (2728...
Miller L, Woodward EM, Campos JR, Squires EL, Troedsson M.The objectives of this study were to (i) verify localization of SP22 on fresh, cooled, and frozen/thawed equine spermatozoa and to (ii) determine SP22 mRNA and protein expression in equine testicular and epididymal tissues. Immunocytochemistry and Western blots were performed on the spermatozoa samples. Northern blots and Western blots were performed on the tissue samples. The immunocytochemistry revealed the presence of SP22 in all samples tested. The fresh spermatozoa stained predominantly over the equatorial segment as did the samples cooled for 1 and 2 days. The samples cooled for 3 days, ...
Cruz Villagrán C, Amelse L, Neilsen N, Dunlap J, Dhar M.Mesenchymal stromal cells (MSCs) are able to differentiate into extramesodermal lineages, including neurons. Positive outcomes were obtained after transplantation of neurally induced MSCs in laboratory animals after nerve injury, but this is unknown in horses. Our objectives were to test the ability of equine MSCs to differentiate into cells of neural lineage in vitro, to assess differences in morphology and lineage-specific protein expression, and to investigate if horse age and cell passage number affected the ability to achieve differentiation. Bone marrow-derived MSCs were obtained from yo...
Jung H, Song H, Yoon M.Putative markers have been discovered and are used to identify and separate certain lineage of spermatogonia. The KIT is a marker for differentiating spermatogonial stem cells in several species including mice and goats. The objectives of this study were (1) to investigate reproductive stage-dependent KIT expression patterns in stallions and (2) to identify spermatogonia subpopulations expressing KIT in stallion testes. To achieve these objectives, testicular samples were obtained during routine field castration of stallions. The reproductive stage of the stallions was classified as pre-pubert...
Duarte CG, Bonilla C, Guimarães G, Machado de Avila RA, Mendes TM, Silva W, Tintaya B, Yarleque A, Chávez-Olórtegui C.In this work, an anti-loxoscelic serum was produced by immunizing horses with a recombinant dermonecrotic protein from Loxosceles intermedia (rLiD1). Anti-rLiD1 antibodies were able to recognize different species of Loxosceles venoms by Western Blot and ELISA. The efficacy of anti-rLiD1 serum against the toxic effects of Loxosceles laeta (Peru) venom was tested, showing that anti-rLiD1 serum can neutralize those effects. This study confirms that recombinant proteins can be good candidates to replace crude venoms for antivenom production.
Yoon M, Jiang J, Chung KH, Roser JF.Insulin-like growth factor plays a paracrine/autocrine role in regulating testicular function in the stallion, but its presence in the equine epididymis remains unknown. The aim of this study was to test the hypothesis that insulin-like growth factor-I (IGF-I) and IGF-I receptor (IGF-IR) are localized in the caput, corpus, and cauda of the epididymis in an age-dependent manner. Immediately after castration, epididymal tissue was fixed, paraffin-embedded, and processed for immunohistochemistry (IHC). Western blot was also performed using equine epididymal extracts to verify the specificity of t...
Tydén E, Tjälve H, Larsson P.Among the cytochrome P450 enzymes (CYP), families 1-3 constitute almost half of total CYPs in mammals and play a central role in metabolism of a wide range of pharmaceuticals. This study investigated gene and protein expression and cellular localisation of CYP1A, CYP2A, CYP2C, CYP2D and CYP2E in equine intestine and liver. Real-time polymerase chain reaction (RT-PCR) was used to analyse gene expression, western blot to examine protein expression and immunohistochemical analyses to investigate cellular localisation. Results: CYP1A and CYP2C were the CYPs with the highest gene expression in the ...
Jung H, Roser JF, Yoon M.Spermatogonial stem cells (SSCs) continuously undergo self-renewal and differentiation to sustain spermatogenesis throughout adulthood in males. In stallions, SSCs may be used for the production of progeny from geldings after cryopreservation and therapy for infertile and subfertile stallions. Undifferentiated cell transcription factor 1 (UTF1) is a putative marker for undifferentiated spermatogonia in humans and rats. The main purposes of this study are to determine the following: 1) changes in the expression pattern of UTF1 at various reproductive stages of stallions, 2) subpopulations of sp...
Lacombe VA, Hinchcliff KW, Devor ST.To characterize insulin-sensitive glucose-transporter (GLUT-4) protein in equine tissues and determine effects of exercise and glucose administration on content of GLUT-4 protein in equine skeletal muscle. Methods: Tissue samples from 9 horses. Methods: Western blot analyses were performed on crude membrane preparations of equine tissues to characterize GLUT-4. In a crossover, randomized study, horses were strenuously exercised for 3 consecutive days and then administered 13.5% glucose or isotonic saline (0.9% NaCl; control) solution, i.v., at similar infusion rates for 12.1 hours. Samples wer...
Borzacchiello G, Mogavero S, De Vita G, Roperto S, Della Salda L, Roperto F.The equine sarcoid is the most common dermatologic neoplasm reported in horses. Bovine papillomavirus (BPV) types 1 and 2 are associated with sarcoids, in which the expression of the major transforming oncoprotein (E5) is often recorded. The transformation activity of the virus is due to the binding of the E5 to the platelet-derived growth factor beta receptor (PDGFbeta-r). In the present study, we show by Western blot in 4 sarcoid samples and 3 normal equine skin samples that the PDGFbeta-r is more phosphorylated in sarcoid tissue than in normal skin (P < .001). Furthermore, the physical i...
Johnson A, Smith R, Saxne T, Hickery M, Heinegård D.Previous experiments have shown that addition of fragmented fibronectin can induce cartilage chondrolysis. In this study we investigated the fate of the collagen- and cell-binding molecules Cartilage oligomeric matrix protein (COMP) and chondroadherin. Methods: Equine articular cartilage explants were stimulated with the C-terminal and the N-terminal heparin-binding fragments of fibronectin respectively, and the conditioned media were analysed by both quantitative (ELISA) and qualitative (mass spectrometry, Western blots) methods. Results: Both COMP and chondroadherin were released in a dose-d...
Klein C, Troedsson MH, Rutllant J.The expression of 12 different aquaporin subtypes in equine endometrium was examined at the mRNA and protein level. Endometrial samples were obtained during anoestrus, oestrus, 8, and 14 days after ovulation in non-pregnant mares, and 14 days after ovulation in pregnant mares. Quantitative PCR revealed a time-dependent pattern for all aquaporin subtypes examined except for AQP10 and 12. AQP3, 5 and 7 showed highest mRNA abundance 8 days after ovulation, while AQP0 and 2 were most abundant at Day 14 of the cycle in non-pregnant mares. At 14 days of pregnancy, AQP1, 4, 8, 9 and 11 displayed ...
Linardi RL, Megee SO, Mainardi SR, Senoo M, Galantino-Homer HL.The limited characterization of equine skin, eye and hoof epithelial stem cell (ESC) and differentiation markers impedes the investigation of the physiology and pathophysiology of these tissues. Objective: To characterize ESC and differentiation marker expression in epithelial tissues of the equine eye, haired skin and hoof capsule. Methods: Indirect immunofluorescence microscopy and immunoblotting were used to detect expression and tissue localization of keratin (K) isoforms K3, K10, K14 and K124, the transcription factor p63 (a marker of ESCs) and phosphorylated p63 [pp63; a marker of ESC tr...
Kupczyk P, Rykala M, Serek P, Pawlak A, Slowikowski B, Holysz M, Chodaczek G, Madej JP, Ziolkowski P, Niedzwiedz A.The endocannabinoid system (ECS) is composed of cannabinoid receptors type 1 (CBR1) and type 2 (CBR2), cannabinoid-based ligands (endogenous chemically synthesized phytocannabinoids), and endogenous enzymes controlling their concentrations. Cannabinoid receptors (CBRs) have been identified in invertebrates and in almost all vertebrate species in the central and peripheral nervous system as well as in immune cells, where they control neuroimmune homeostasis. In humans, rodents, dogs, and cats, CBRs expression has been confirmed in the skin, and their expression and tissue distribution become di...
Saville WJ, Reed SM, Granstrom DE, Hinchcliff KW, Kohn CW, Wittum TE, Stamper S.To determine the seroprevalence of serum antibodies to Sarcocystis neurona in horses residing in Ohio. Methods: Prevalence survey. Methods: Serum from samples from 1,056 horses. Serum was collected on every 36th sample submitted to the Ohio State Diagnostic Laboratory for testing for equine infectious anemia. Methods: Serum was frozen at -80 C and analyzed for antibodies to S neurona, using a western blot. Information regarding blood sample collection, age, breed, sex, and geographic location was recorded for each horse. Data were analyzed, using multivariable logistic regression. Results: Hor...
Cheadle MA, Lindsay DS, Rowe S, Dykstra CC, Williams MA, Spencer JA, Toivio-Kinnucan MA, Lenz SD, Newton JC, Rolsma MD, Blagburn BL.An IFAT was used to determine the prevalence of Neospora-specific IgG antibodies in serum from Alabama horses. Serum samples (n = 536) were from asymptomatic horses routinely submitted for equine infectious anaemia virus infection testing. We also subjected a 13-year-old horse with CNS disease to necropsy examination for isolation and in vitro cultivation of protozoal organisms. In antemortem tests, this horse was positive for antibodies to Neospora sp. in the IFAT and western immunoblot. Results of the prevalence survey indicated that IgG antibodies to Neospora were present in 62 (11.5%) of t...
Busschers E, Holt JP, Richardson DW.To determine effects of interleukin (IL)-1 beta and glucocorticoids on total glycosaminoglycan (GAG) loss and aggrecanase-mediated matrix degradation in equine cartilage. Methods: Cartilage from 24 equine cadavers free of sepsis and musculoskeletal disease. Methods: Effects of IL-1 beta, IL-1 beta with glucocorticoids (dexamethasone and triamcinolone, 10(-6) and 10(-7)M), and glucocorticoids alone on degradation of equine articular and nasal cartilage explants were assessed by measuring GAG release in media and GAG content in cartilage. Aggrecanase-mediated cleavage within the interglobular do...
Doty A, Buhi WC, Benson S, Scoggin KE, Pozor M, Macpherson M, Mutz M, Troedsson MH.Equine spermatozoa induce a uterine inflammatory response characterized by a rapid, transient influx of polymorphonuclear neutrophils (PMNs). Seminal plasma proteins have been shown to modulate the interaction between spermatozoa and PMNs, but a specific protein responsible for this function has not been identified. The objective of this study was to isolate and identify a protein in equine seminal plasma that suppresses binding between spermatozoa and PMNs. Seminal plasma was pooled from five stallions, and proteins were precipitated in 60% (w/v) ammonium sulfate and dialyzed (3500 MW cutoff)...
van der Meide NM, Meulenbroeks C, van Altena C, Schurink A, Ducro BJ, Wagner B, Leibold W, Rohwer J, Jacobs F, van Oldruitenborgh-Oosterbaan MM....Insect bite hypersensitivity (IBH) is an allergic dermatitis in horses caused by the bites of Culicoides species. The aim of the present study was to evaluate the applicability of whole body extracts of C. obsoletus (the main species found feeding on horses in the Netherlands), C. nubeculosus (rarely found in The Netherlands) and C. sonorensis (typical for North America) for diagnosis of IBH in horses in The Netherlands. Blood and serum samples of 10 clinically confirmed IBH affected and 10 healthy control horses were used to evaluate the IgE titers (ELISA) against the Culicoides whole body ex...
Mort JS, Beaudry F, Théroux K, Emmott AA, Richard H, Fisher WD, Lee ER, Poole AR, Laverty S.To characterize the initial events in the cleavage of type II collagen mediated by cathepsin K and demonstrate the presence of the resulting products in human and equine articular osteoarthritic cartilage. Equine type II collagen was digested with cathepsin K and the cleavage products characterized by mass spectrometry. Anti-neoepitope antibodies were raised against the most N-terminal cleavage products and used to investigate the progress of collagen cleavage, in vitro, and the presence of cathepsin K-derived products in equine and human osteoarthritic cartilage. Six cathepsin K cleavage sit...
Waller AP, Huettner L, Kohler K, Lacombe VA.Although insulin resistance (IR) has been increasingly recognized in horses, a clear understanding of its pathophysiology is lacking. The purpose of the present study was to determine the early pathologic changes in IR horses by characterizing alterations in proteins that play key roles in innate immunological responses and inflammatory pathways, and by identifying potential links with glucose transport and insulin signaling. Visceral (VIS) and subcutaneous (SC) adipose tissue and skeletal muscle (SM) biopsies were collected from horses, which were classified as insulin-sensitive (IS) or IR ba...
Rout ED, Webb TL, Laurence HM, Long L, Olver CS.Evaluation of erythrocyte regeneration in horses is challenging, as they do not release reticulocytes into the peripheral blood. This study investigated transferrin receptor 1 (TfR1) expression in exosomes as a noninvasive method of characterising the regenerative response in anaemic horses. Objective: To quantify TfR1 in ultraprecipitate of serum in horses before and after phlebotomy-induced anaemia, and to identify exosomes as the source of TfR1. The hypothesis was that serum exosomal TfR1 expression would increase during a regenerative response. Methods: Experimental model of anaemia. Metho...
Jaworska J, Janowski T.Retained fetal membranes (RFM) is a prevalent postpartum disease in mares. During parturition, various proinflammatory cytokines are released in the placenta, which further facilitates uterine contractions, expulsion of the fetus and fetal membranes. We hypothesized that an altered inflammatory response in mares results in retained fetal membranes. Samples of the endometrium and the allantochorion were collected from 15 mares with RFM and from 29 control mares within 2 h of foal delivery. Next, the mRNA expression of IL-1β, IL-6 and TNFα proteins was analyzed by Real Time PCR and Western b...
Adreani CM, Li ZB, Lehar M, Southwood LL, Habecker PL, Flint PW, Parente EJ.The myosin heavy chain (MHC) composition of a given muscle determines the contractile properties and, therefore, the fiber type distribution of the muscle. MHC isoform expression in the laryngeal muscle is modulated by neural input and function, and it represents the cellular level changes that occur with denervation and reinnervation of skeletal muscle. The objective of this study was to evaluate the pattern of MHC isoform expression in laryngeal muscle harvested from normal cadavers and cadavers with naturally occurring left laryngeal hemiplegia secondary to recurrent laryngeal neuropathy. L...
Skiöldebrand E, Heinegård D, Eloranta ML, Nilsson G, Dudhia J, Sandgren B, Ekman S.The aim of the present study was to correlate the levels of COMP and aggrecan as indicators of tissue damage, in synovial fluid (sf) from carpal joints of acutely lame racehorses, with macroscopical lesions of articular cartilage (OA), osteochondral fractures and ligament tears found at arthroscopy. Sixty-three lame horses [49 Standardbred trotters (STB) and 14 Thoroughbreds (TB)] in conventional training and racing that underwent arthroscopy of their middle carpal or radiocarpal joints were included in the study. Intact as well as fragmented COMP and aggrecan released into the synovial fluid ...
Böse R, Daemen K.Babesia caballi-infected or normal equine erythrocytes were solubilized in sodium dodecyl sulfate (SDS) buffer and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Antigens were allowed to react with sera from horses experimentally or field-infected with B. caballi and with sera from non-infected horses. Major babesial antigens recognized by immune sera had apparent mol. wts of 141, 112, 70, 50, 48, 34, and 30 kDa. The polypeptides at 50 and 48 kDa were recognized earliest and throughout infection, but also weakly by 3/100 equine sera tested negative and 1/33...
Ikadai H, Nagai A, Xuan X, Igarashi I, Tsugihiko K, Tsuji N, Oyamada T, Suzuki N, Fujisaki K.Antibodies to Babesia caballi and Babesia equi were examined on a total of 2,019 horse serum samples that had been collected in 1971-1973 by the National Institute of Animal Health by enzyme-linked immunosorbent assay (ELISA) using recombinant proteins and by Western-blot analysis. Based on the criterion for positivity by ELISA, 5.4% (109/2,019) and 2.2% (44/2,019) had antibodies against B. caballi and B. equi, respectively. The ELISA-positive sera were further examined by Western blot; 30/109 for B. caballi and 2/ 44 for B. equi were positive for native B. caballi or B. equi, but none of them...
Kwon S, Gewirtz AT, Hurley DJ, Robertson TP, Moore JN, Vandenplas ML.As sentinel cells of the innate immune system, neutrophils and mononuclear phagocytes use specific TLRs to recognize the conserved molecular patterns that characterize microbes. This study was performed to compare the responses of equine neutrophils and mononuclear phagocytes to LPS and flagellin, components of bacteria that are recognized by TLR4 and TLR5, respectively. Neutrophils and mononuclear phagocytes isolated from healthy horses were incubated in vitro with LPS, flagellin, or pronase-inactivated flagellin in the presence or absence of polymyxin B. Production of reactive oxygen species...
Gibson KE, Pastenkos G, Moesta S, Rikihisa Y.Neorickettsia risticii is the Gram-negative, obligate, and intracellular bacterial pathogen responsible for Potomac horse fever (PHF): an important acute systemic disease of horses. N. risticii surface proteins, critical for immune recognition, have not been thoroughly characterized. In this paper, we identified the 51-kDa antigen (P51) as a major surface-exposed outer membrane protein of older and contemporary strains of N. risticii through mass spectrometry of streptavidin-purified biotinylated surface-labeled proteins. Western blot analysis of sera from naturally-infected horses demonstrate...
Dangoudoubiyam S, Oliveira JB, Víquez C, Gómez-García A, González O, Romero JJ, Kwok OC, Dubey JP, Howe DK.Serum samples from 315 horses from Costa Rica, Central America, were examined for the presence of antibodies against Sarcocystis neurona, Neospora spp., and Toxoplasma gondii by using the surface antigen (SAG) SnSAG2 enzyme-linked immunosorbent assay (ELISA), the NhSAG1 ELISA, and the modified agglutination test, respectively. Anti- S. neurona antibodies were found in 42.2% of the horses by using the SnSAG2 ELISA. Anti- Neospora spp. antibodies were found in only 3.5% of the horses by using the NhSAG1 ELISA, and only 1 of these horses was confirmed seropositive by Western blot. Antibodies to T...
Fortier LA, Miller BJ.During aging, chondrocytes become unresponsive to insulin-like growth factor-I (IGF-I). This study examined the role of Cdc42 (cell-division-cycle 42) in IGF-I signaling during aging. Experiments were performed using cartilage and chondrocytes isolated from horses ages 1 day-25 years. Northern analysis was used to examine expression of the small GTPases Cdc42, Rac, and RhoA. Western analysis was utilized to assess total Cdc42 (GTP + GDP-bound); active, GTP-Cdc42 was assessed using a pulldown assay with Western analysis. GTP-Cdc42 was also measured following IGF-I treatment. Gene expression for...
Egito AS, Miclo L, López C, Adam A, Girardet JM, Gaillard JL.The equine alpha(s1)- and beta-caseins (CN) were purified by chromatography on DEAE-cellulose and by reversed-phase HPLC. The alpha(s1)-, beta-, and kappa-CN were characterized either by monodimensional urea-PAGE or sodium dodecylsulfate (SDS)-PAGE or by bidimensional electrophoresis. Kappa-casein was characterized after electrophoresis by glycoprotein-specific staining. To identify alpha(s1)-CN without ambiguity, internal sequences were determined after trypsin or chymosin digestion of purified alpha(s1)-CN. These sequences, that could be estimated to correspond to 62% of the full protein, pr...
Lannergård J, Flock M, Johansson S, Flock JI, Guss B.Streptococcus equi subsp. equi is the causative agent of strangles, a disease of the upper respiratory tract in horses. The initiation of S. equi subsp. equi infection is likely to involve cell surface-anchored molecules mediating bacterial adhesion to the epithelium of the host. The present study describes the cloning and characterization of FNEB, a fibronectin-binding protein with cell wall-anchoring motifs. FNEB can thus be predicted as cell surface located, contrary to the two previously characterized fibronectin-binding proteins in S. equi subsp. equi, FNE and SFS. Assays of antibody tite...
Fink C, Hembes T, Brehm R, Weigel R, Heeb C, Pfarrer C, Bergmann M, Kressin M.In the glandular stomach, gap junctional intercellular communication (GJIC) plays an important role in the gastric mucosal defense system, and loss of GJIC is associated with ulcer formation. In spite of the high incidence of gastric ulcers in horses, particularly at pars nonglandularis, the presence of gap junctions in the equine stomach has not yet been studied. The objective was to obtain basic data on the distribution of gap junction protein connexin 32 (Cx32) in the different regions of normal equine gastric mucosa. Samples of mucosa were taken from seven horses at cardiac, fundic, and py...
McGorum BC, Pirie RS, Eaton SL, Keen JA, Cumyn EM, Arnott DM, Chen W, Lamont DJ, Graham LC, Llavero Hurtado M, Pemberton A, Wishart TM.Equine grass sickness (EGS) is an acute, predominantly fatal, multiple system neuropathy of grazing horses with reported incidence rates of ∼2%. An apparently identical disease occurs in multiple species, including but not limited to cats, dogs, and rabbits. Although the precise etiology remains unclear, ultrastructural findings have suggested that the primary lesion lies in the glycoprotein biosynthetic pathway of specific neuronal populations. The goal of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS. Here, we use a bottom-up approach be...
Parma AE, Sanz ME, Lucchesi PM, Mazzonelli J, Petruccelli MA.A protein epitope which is involved in an antigenic relationship between equine ocular tissues and Leptospira interrogans was detected in homogenates of the bacterium. The antigenic determinant was harboured on a peptide structure which was shown to be sensitive to the action of denaturing and reducing agents by means of Western blotting. The outer surface of the leptospires appeared to be free of this epitope as was proved by dot-blot and electron microscopic studies.
González-Fernández L, Sánchez-Calabuig MJ, Calle-Guisado V, García-Marín LJ, Bragado MJ, Fernández-Hernández P, Gutiérrez-Adán A....A repeatable protocol for equine in vitro fertilization (IVF) has remained elusive. This is likely, in part, due to suboptimal composition of capacitation or IVF media that are currently in use. Hence, we aimed to analyse the metabolome of equine oviductal fluid (OF) at the pre- (PRE) and immediate post-ovulatory (PST) stages using proton magnetic resonance spectroscopy (H NMR). Oviductal fluid from eight PRE and six PST mares were used to prepare a total of five samples per group. A total of 18 metabolites were identified. The five metabolites with the highest concentrations in the OF sample...
Kankavi O, Ata A, Gungor O.The presence of surface-active material in the lung alveolus has been known for several decades as being essential for normal lung function. Surfactant is essential for reducing the surface tension at the alveolar air-liquid interface. Pulmonary surfactant is composed of 90% lipids and 10% proteins. There are four non-serum proteins surfactant protein-A (SP-A), surfactant protein-B (SP-B), surfactant protein-C (SP-C) and surfactant protein-D (SP-D) named in chronologic order of discovery. Lung SP-A and SP-D belong to a family of collagen-containing C-type lectin family called collectins. The h...
