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.
Buss DG, Sharma A, Giuliano EA, Mohan RR.Mitomycin C (MMC) is used clinically to treat corneal scarring in human patients. We investigated the safety and efficacy of MMC to treat corneal scarring in horses by examining its effects at the early and late stages of disease using an in vitro model. Methods: An in vitro model of equine corneal fibroblast (ECF) developed was used. The ECF or myofibroblast cultures were produced by growing primary ECF in the presence or absence of transforming growth factor beta-1 (TGFbeta1) under serum-free conditions. The MMC dose for the equine cornea was defined with dose-dependent trypan blue exclusion...
Albrizio M, Lacalandra GM, Micera E, Guaricci AC, Nicassio M, Zarrilli A.Opioid receptors and endogenous opioid peptides act not only in the control of nociceptive pathways, indeed several reports demonstrate the effects of opiates on sperm cell motility and morphology suggesting the importance of these receptors in the modulation of reproduction in mammals. In this study we investigated the expression of delta opioid receptors on equine spermatozoa by western blot/indirect immunofluorescence and its relationship with sperm cell physiology. Methods: We analyzed viability, motility, capacitation, acrosome reaction and mitochondrial activity in the presence of naltri...
Abraham G, Shibeshi W, Ungemach FR.Responses and functions of airway epithelial cells are stimulated by β₂-agonists via the β₂-adrenergic receptors (β₂-ARs)-G(s)-protein-cAMP-system, thus, affecting airway inflammation such as in asthma and equine recurrent airway obstruction (RAO). Though horses can be used as large animal model for human asthma, evaluation of the expression and functions of the β-AR system in primary equine airway epithelial cells has not been yet carried out. Thus, for the first time, we determined the β-AR density and subtype distribution by [¹²⁵I]-iodocyanopindolol (ICYP) binding, examined ...
Yoon MJ, Berger T, Roser JF.The insulin-like growth factor-I (IGF-I) is a key regulator of reproductive functions. IGF-I actions are primarily mediated by IGF-IR. The main objective of this research was to evaluate the presence of IGF-I and IGF-I Receptor (IGF-IR) in stallion testicular tissue. The hypotheses of this study were (i) IGF-I and IGF-IR are present in stallion testicular cells including Leydig, Sertoli, and developing germ cells, and (ii) the immunolabelling of IGF-I and IGF-IR varies with age. Testicular tissues from groups of 4 stallions in different developmental ages were used. Rabbit anti-human polyclona...
Skotarek SL, Colwell DD, Goater CP.Accurate diagnosis of parasites within individual hosts remains a difficult task. Incorrect and/or inaccurate diagnosis restricts the potential for targeted treatment of individuals and limits our understanding of key epidemiological characteristics of potential pathogens of domestic stock. In this study, we compared the specificity and sensitivity of four diagnostic methods for determination of the presence and intensity of the cestode Anoplocephala perfoliata in horses. Over 400 horses from an abattoir in south-central Alberta were evaluated for the presence and number of cestodes. Thirty on...
Schaffartzik A, Marti E, Crameri R, Rhyner C.Insect bite hypersensitivity (IBH) is an IgE-mediated seasonal dermatitis of the horses associated with bites of Simulium (black fly) and Culicoides (midge) species. Although cross-reactivity between Simulium and Culicoides salivary gland extracts has been demonstrated, the molecular nature of the allergens responsible for the observed cross-reactivity remains to be elucidated. In this report we demonstrate for the first time in veterinary medicine that a homologous allergen, present in the salivary glands of both insects, shows extended IgE cross-reactivity in vitro and in vivo. The cDNA sequ...
Echigoya Y, Okabe H, Itou T, Endo H, Sakai T.Muscle glycogen synthase (GYS1) is the rate-limiting enzyme in glycogen synthesis, and its activity is regulated by the phosphorylation states of certain amino acid residues encoded by the GYS1 gene. In the present study, the authors molecularly characterized the full-length equine GYS1 (eGYS1) cDNA and found that it contains a less common polyadenylation signal (AATACA). An amino acid alignment with other mammalian GYS1 showed that the phosphorylation sites in eGYS1 are completely conserved. Genomic DNA analysis revealed that the equine-specific substitutions (Glu 16 Asp and Ala 252 Thr) were...
Zerpa H, Berhane Y, Woodcock H, Elliott J, Bailey SR.A decrease in environmental temperature can directly affect the contractility of cutaneous vasculature, mediated in part by alpha(2)-adrenoceptors. Most of the cellular mechanisms underlying the cooling-enhanced contractility to alpha(2)-adrenoceptor agonists have been reported in cutaneous arteries but little information is available on cutaneous veins. To investigate the cellular mechanisms associated with the cooling-enhanced contraction to UK-14304 (alpha(2)-adrenoceptor agonist), isolated equine digital veins (EDVs) were studied at 30 degrees C and 22 degrees C. The effects of inhibitors ...
Visser MB, Pollitt CC.Most research to date involving laminins and extracellular matrix protein function in both normal and pathological conditions involves in vitro culture of keratinocytes. Few methods are established to allow for prolonged propagation of keratinocytes from equine tissues, including the hoof lamellae. In this study we modified cell isolation and culture techniques to allow for proliferation and sub-culturing of equine lamellar keratinocytes. Additionally, the production and processing of extracellular matrix molecules by skin and lamellar keratinocytes were studied. Results: Physical and proteoly...
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...
Pickles KJ, Brooks AC, Rickards KJ, Cunningham FM.N-terminal peptides derived from the anti-inflammatory peptide, annexin-1, inhibit neutrophil function but can also induce pro-inflammatory effects. Although equine annexin-1 has been sequenced, its cellular expression and properties have not been reported. This study has examined whether annexin-1 is present in equine leucocytes and how the N-terminal peptide, Ac2-26, affects equine neutrophil superoxide production. Annexin-1 expression in equine neutrophils and mononuclear cells and the ability of Ac2-26 to activate neutrophil p42/44 MAPK were determined by immunoblotting. Equine neutrophil ...
Milillo P, Traversa D, Elia G, Otranto D.Larvae of Rhinoestrus spp. (Diptera, Oestridae) infect nasal and sinus cavities of horses, causing a nasal myiasis characterized by severe respiratory distress. Presently, the diagnosis of horse nasal botfly relies on the observation of clinical signs, on the post mortem retrieval of larvae or on molecular assays performed using pharyngeal swabs. The present study was carried out to characterize larval somatic proteins and salivary glands of Rhinoestrus spp. in a preliminary assessment towards the immunodiagnosis of equine rhinoestrosis. Out of the 212 necropsied horses 13 were positive for th...
Ramery E, Fievez L, Fraipont A, Bureau F, Lekeux P.The long pentraxin 3 (PTX3) plays an important role in host defence and its over-expression may contribute to airway injury. The aim of the present study was therefore to characterize in more detail PTX3 and its expression in the horses' airway. Six healthy horses and six horses affected by recurrent airway obstruction (R.A.O.) were submitted to a dusty environment challenge. PTX3 DNA and cDNA were cloned and sequenced. PTX3 expression was evaluated by RT-qPCR, Western blotting and immunohistochemistry in bronchoalveolar lavage fluid (BALF) cells, BALF supernatant and bronchial epithelial cell...
Cook VL, Jones Shults J, McDowell MR, Campbell NB, Davis JL, Marshall JF, Blikslager AT.To investigate effects of lidocaine hydrochloride administered IV on mucosal inflammation in ischemia-injured jejunum of horses treated with flunixin meglumine. Methods: 24 horses. Methods: Horses received saline (0.9% NaCl) solution (SS; 1 mL/50 kg, IV [1 dose]), flunixin meglumine (1 mg/kg, IV, q 12 h), lidocaine (bolus [1.3 mg/kg] and constant rate infusion [0.05 mg/kg/min], IV, during and after recovery from surgery), or both flunixin and lidocaine (n = 6/group). During surgery, blood flow was occluded for 2 hours in 2 sections of jejunum in each horse. Uninjured and ischemia-injured jejun...
Zipplies JK, Hauck SM, Schoeffmann S, Amann B, van der Meijden CH, Stangassinger M, Ueffing M, Deeg CA.Equine recurrent uveitis (ERU) is an incurable disease affecting the inner eye that leads to blindness, through activated T cells that pass the blood-retinal barrier and destroy the retina. Serum markers are a desirable choice for monitoring development of disease, as serum is easy accessible and the markers could serve to predict the beginning of disease or an imminent relapse. Methods: In this study, serum proteomes (depleted of high-abundance serum proteins) of horses with ERU and healthy controls were compared with the 2-D DIGE (two-dimensional gel electrophoresis) technique to identify di...
Cook VL, Meyer CT, Campbell NB, Blikslager AT.To determine whether treatment of horses with firocoxib affects recovery of ischemic-injured jejunum, while providing effective analgesia. Methods: 18 horses. Methods: Horses (n = 6 horses/group) received saline (0.9% NaCl) solution (1 mL/50 kg, IV), flunixin meglumine (1.1 mg/kg, IV, q 12 h), or firocoxib (0.09 mg/kg, IV, q 24 h) before 2 hours of jejunal ischemia. Horses were monitored via pain scores and received butorphanol for analgesia. After 18 hours, ischemic-injured and control mucosa were placed in Ussing chambers for measurement of transepithelial resistance and permeability to lipo...
Ortega Ferrusola C, González Fernández L, Macías García B, Salazar-Sandoval C, Morillo Rodríguez A, Rodríguez Martinez H, Tapia JA, Peña FJ.The ability of stallion spermatozoa to produce nitric oxide (NO) before (fresh) and after freezing and thawing (FT) was evaluated by means of flow cytometry after loading the sperm suspension with the probe, 4,5-diaminofluorescenin diacetate. The presence of NO synthase (NOS) was investigated by Western blotting using anti-NOS1, anti-NOS3, or anti-universal NOS antibodies (Abs). While NO was detected both in fresh and FT sperm suspensions, its production increased after cryopreservation only when egg yolk was removed from the extender. Anti-NOS1 Ab intensively labeled a single band with an app...
van den Burg MM, Eizema K, de Graaf-Roelfsema E, van Breda E, Wijnberg ID, van der Kolk JH, Everts ME.To investigate the effects of acute exercise and long-term training on Na(+),K(+)-ATPase content, mRNA isoforms, and protein concentration in equine muscle. Methods: 6 Standardbreds. Methods: Horses performed a bout of exercise on a treadmill before and after 18 weeks of combined interval and endurance training. Muscle biopsy specimens were obtained from vastus lateralis muscle (VLM) and pectoralis descendens muscle (PDM) before and after exercise. The Na(+),K(+)-ATPase content, mRNA isoforms, and protein concentrations were determined by use of [(3)H]ouabain binding, real-time PCR assay, and ...
Duehlmeier R, Hacker A, Widdel-Bigdely A, von Engelhardt W, Sallmann HP.Glucose homeostasis depends on insulin-regulated glucose uptake in the skeletal muscles and fat tissues via glucose transporter (GLUT) 4 translocation into cellular plasma membranes. The present study sought to elucidate GLUT4 expression, GLUT1 and GLUT4 translocation and glucose uptake in the skeletal muscles of Shetland ponies. Semitendinosus muscle explants were removed by open muscle biopsy from six Shetland pony geldings under general anaesthesia. The expression of GLUT4 was analysed by measuring muscle crude membrane (CM) GLUT4 protein contents. To determine the insulin-stimulated GLUT t...
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...
Matyjaszek SA, Morton AJ, Freeman DE, Grosche A, Polyak MM, Kuck H.To examine the effects of flunixin meglumine (FM) on recovery of colonic mucosa from experimentally induced ischemia in horses. Methods: 14 research horses. Methods: Ischemia was induced in the colons of anesthetized horses for 2 hours. Afterward, horses received saline (0.9% NaCl) solution (12 mL, IV, q 12 h; n = 7) or FM (1.1 mg/kg, IV, q 12 h; 7) and were allowed to recover for 18 hours after termination of the ischemic event. Postoperative pain scores were recorded every 4 hours throughout the recovery period. At the end of the recovery period, horses were anesthetized, and ischemic and no...
González-Fernández L, Ortega-Ferrusola C, Macias-Garcia B, Salido GM, Peña FJ, Tapia JA.Protein tyrosine kinases have important roles in spermatozoa; however, little is known about the presence and regulation in these cells of their counterparts in signaling, namely, protein tyrosine phosphatases (PTPs) and dual-specificity phosphatases (DSPs). The objectives of the present study were to identify PTPs and DSPs in boar, stallion, and dog spermatozoa; to characterize their subcellular distribution; and to investigate the roles of tyrosine phosphatases in maintenance of protein tyrosine phosphorylation level and in sperm motility. Using Western blotting with specific antibodies in b...
Hendricks KE, Hansen PJ.Apoptosis is common during spermatogenesis. Here, it was tested whether apoptosis could be induced in sperm after ejaculation. There were several lines of evidence to indicate that sperm are resistant to induction of apoptosis. First, incubation of bull sperm at temperatures characteristic of normothermia (38.5 degrees C) or heat shock (40 and 41 degrees C) for 4h did not increase the proportion of sperm positive for the TUNEL reaction. There was also no reduction in mitochondrial polarity caused by exposure to 40 or 41 degrees C. Incubation at 38.5 degrees C (least-squares mean+/-SEM=4.0+/-1....
Roelfstra L, Deeg CA, Hauck SM, Buse C, Membrez M, Betschart B, Pfister K.Little information is available on the immunological aspect of parasitic Gasterophilus intestinalis (Diptera, Oestridae) larvae causing horse gastric myiasis. The objectives of this research were to analyze the protein content of larval crude extracts of the migrating second and third larvae (L2 and L3) of G. intestinalis in order to characterize the immune response of horses. Results: The proteomic profile of L2 and L3, investigated by using one and two dimensional approaches, revealed a migration pattern specific to each larval stage. Furthermore, Western blots were performed with horse sera...
Black WD, Hartley CA, Ficorilli NP, Studdert MJ.Equine rhinitis B virus (ERBV), genus Erbovirus, is most closely related to the Cardiovirus genus in the family Picornaviridae. The structural proteins (VP1-4) of erboviruses are not well described, but are predicted by sequence to be 35, 29, 26 and 7 kDa. Methods for the purification of cardioviruses (polyethylene glycol, trypsin treatment) were used to characterise the structural proteins of ERBV1. Only one of the virus proteins detected was an expected molecular mass, and this 26 kDa protein was identified as VP3 by N-terminal amino acid sequencing. N-terminal sequencing of the 56 and a 29 ...
Bucci D, Isani G, Spinaci M, Tamanini C, Mari G, Zambelli D, Galeati G.Spermatozoa, as other eukaryotic cells, need hexoses to produce energy to maintain membrane homeostasis, to move along the female genital tract and to carry the male genome to the female gamete. GLUTs are a family of proteins that permit and improve the passive transport of hexoses inside cells. This study was aimed at investigating the presence and localization of GLUTs 1, 2, 3 and 5 in boar, stallion and dog spermatozoa by both immunofluorescence and western blotting. GLUTs exhibited a peculiar distribution along the sperm cell depending on the isoforms considered, the hexose they transport ...
Dyer J, Al-Rammahi M, Waterfall L, Salmon KS, Geor RJ, Bouré L, Edwards GB, Proudman CJ, Shirazi-Beechey SP.Experimental and epidemiological evidence suggests that consumption of hydrolyzable carbohydrate, hCHO (grain), by horses is an important risk factor for colic, a common cause of equine mortality. It is unknown whether the small intestinal capacity to digest hCHO and/or to absorb monosaccharides is limiting, or even if horses can adapt to increased carbohydrate load. We investigated changes in the brush-border membrane carbohydrate digestive enzymes and glucose absorptive capacity of horse small intestine in response to increased hCHO. Expression of the Na(+)/glucose co-transporter, SGLT1, was...
Hobson-Peters J, Toye P, Sánchez MD, Bossart KN, Wang LF, Clark DC, Cheah WY, Hall RA.Using a monoclonal antibody directed to domain I of the West Nile virus (WNV) envelope (E) protein, we identified a continuous (linear) epitope that was immunogenic during WNV infection of horses. Using synthetic peptides, this epitope was mapped to a 19 aa sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through ...
Bailey SR, Adair HS, Reinemeyer CR, Morgan SJ, Brooks AC, Longhofer SL, Elliott J.The link between the fermentation of carbohydrate in the equine large intestine and the development of acute laminitis is poorly understood. Absorption of endotoxin (lipopolysaccharide; LPS) into the plasma has been observed in one experimental model of laminitis, but does not cause laminitis when administered alone. Thus, the potential role of endotoxin is unclear. Platelet activation has previously been demonstrated in the developmental stage of laminitis. Equine platelets are more sensitive than leukocytes to activation by endotoxin, and can be activated directly by LPS in the low pg/ml ran...
Brooks AC, Menzies-Gow NJ, Wheeler-Jones C, Bailey SR, Cunningham FM, Elliott J.The endothelium plays a major role in the pathogenesis of endotoxemia. Binding of endotoxin (lipopolysaccharide; LPS) to endothelium initiates a range of signalling events, including activation of p38 mitogen activated protein kinases (MAPKs) that are involved in the initiation of inflammatory responses. In the present study we have examined whether clinically relevant concentrations of LPS can activate p38 MAPK in equine endothelial cells and have investigated the role of the kinase in neutrophil adhesion and mediator release. Cultured equine digital vein endothelial cells (EDVEC) were expose...
Daraban Bocaneti F, Altamura G, Corteggio A, Tanase OI, Dascalu MA, Pasca SA, Hritcu O, Mares M, Borzacchiello G.Bovine papillomaviruses -1/-2 (BPVs) are small non-enveloped double-stranded DNA viruses able to infect the skin of bovids and equids, causing development of neoplastic lesions such as bovine cutaneous fibropapillomas and equine sarcoid. Matrix metalloproteinases (MMPs) are a group of zinc-dependent endopeptidases that degrade basal membrane and extracellular matrix, whose function is essential in physiological processes such as tissue remodeling and wound healing. MMPs activity is finely regulated by a balancing with expression of tissue inhibitors of MMPs (TIMPs), a process that is impaired ...
Xiao C, Liu Y, Jiang Y, Magoffin DE, Guo H, Xuan H, Wang G, Wang LF, Tu C.Four monoclonal antibodies (mAbs) were generated by immunizing BALB/C mice with recombinant nucleocapsid protein (N) of Nipah virus (NiV) and Hendra virus (HeV) expressed in E. coli. Two mAbs each were obtained for the HeV N and NiV N, respectively. All four mAbs displayed specific reactivity with the recombinant N proteins of both viruses by western blot, which was further confirmed by immunofluorescent antibody assay using fixed insect cells infected with recombinant baculoviruses expressing either the HeV or NiV N protein. Epitope mapping using a 12-mer random peptide phage display library ...
Wang LF, Gould AR, Selleck PW.Full-length cDNA clones coding for the matrix (M) and fusion (F) proteins of equine morbillivirus (EMV) were isolated by RT-PCR, and expressed in Escherichia coli using two different expression systems. Western blot analysis indicated that the M and F proteins, expressed either by itself or as fusion proteins with glutathione S-transferase (GST), were insoluble and degraded after expression. Analysis of the degradation pattern of recombinant M protein suggested that the N-terminus of the matrix protein might be more stable and antigenic than the C-terminal region. Therefore a third system was ...
Rossi HS, Koho NM, Ilves M, Rajamäki MM, Mykkänen AK.OBJECTIVE To examine whether expression of extracellular matrix metalloproteinase inducer (EMMPRIN) can be detected in equine lungs and whether it correlates with matrix metalloproteinase (MMP)-2 and -9 expression in bronchoalveolar lavage fluid (BALF) of horses with chronic inflammation of the lungs (ie, lower airway inflammation [LAI]). ANIMALS 29 horses with signs of chronic respiratory tract disease, which were classified as the LAI (n = 17) and LAI with respiratory distress (RDLAI [12]) groups, and 15 control horses. PROCEDURES BALF, tracheal aspirate, and blood samples were obtained, and...
Weidmeyer CE, Solter PF.The Incstar(R) SPQ II human haptoglobin (Hpt) (Incstar Corporation, Stillwater, MN) immunoturbidimetric assay was validated for the determination of serum and plasma Hpt concentrations in dogs and horses. The anti-human Hpt antiserum supplied with the assay, displayed monospecificity to both dog and horse serum Hpt by immunoelectrophoresis and Western blotting techniques. The automated immunoturbidimetric assay results correlated well with the cyanmethemoglobin binding assay (r=0.953 for canine serum and r=0.941 for equine serum), and had excellent precision at both high and low serum Hpt conc...
Bourebaba L, Kępska M, Qasem B, Zyzak M, Łyczko J, Klemens M, Mularczyk M, Marycz K.Equine metabolic syndrome (EMS) is a steadily growing endocrine disorder representing a real challenge in veterinary practice. As a multifactorial condition, EMS is characterized by three main metabolic abnormalities including insulin resistance, increased adiposity or obesity and hoof laminitis. Adipose tissue dysfunction is recognized as a core pathophysiological determinant of EMS, as it strongly participates to lipotoxicity and systemic metaflammation, both of which have been closely linked to the development of generalized insulin resistance. Besides, sex hormone binding globulin (SHBG) i...
Cohen ND, Heck FC, Heim B, Flad DM, Bosler EM, Cohen D.Four hundred sixty-nine serum samples were obtained from horses admitted to the internal medicine service of the Texas Veterinary Medical Center between Jan 1 and Dec 31, 1990. Serum samples were tested by ELISA for antibody to Borrelia burgdorferi. Of these 469 samples, 1 (0.2%) was repeatedly seropositive for the organism by ELISA. Confirmatory testing by protein immunoblot was negative. The observed seroprevalence was 0%; the upper bound of the 95% confidence interval was 0.6%. These findings indicate the evidence of infection with B burgdorferi is presently uncommon in horses in central Te...
Misumi K, Vilim V, Clegg PD, Thompson CC, Carter SD.This study was designed to assay cartilage oligomeric matrix protein (COMP) in equine synovial fluids and to compare the concentration in synovial fluids from normal horses with joint diseased horses. The relationship between the COMP degradation and the matrix metalloproteinase activity in synovial fluids was also investigated. Methods: Using COMP antigen prepared from equine articular cartilage and murine monoclonal antibody (12C4) raised against human COMP, an inhibition ELISA was developed. COMP in equine synovial fluids from normal and diseased joints was quantified. Metalloproteinase act...
Mackay RJ, Tanhauser ST, Gillis KD, Mayhew IG, Kennedy TJ.To evaluate the effect of intermittent oral administration of ponazuril on immunoconversion against Sarcocystis neurona in horses inoculated intragastrically with S neurona sporocysts. Methods: 20 healthy horses that were seronegative for S neurona-specific IgG. Methods: 5 control horses were neither inoculated with sporocysts nor treated. Other horses (5 horses/group) each received 612,500 S neurona sporocysts via nasogastric tube (day 0) and were not treated or were administered ponazuril (20 mg/kg, PO) every 7 days (beginning on day 5) or every 14 days (beginning on day 12) for 12 weeks. Bl...
Tachibana Y, Nakano Y, Nagaoka K, Kikuchi M, Nambo Y, Haneda S, Matsui M, Miyake Y, Imakawa K.Despite enormous efforts, biochemical and molecular mechanisms associated with equine reproduction, particularly processes of pregnancy establishment, have not been well characterized. Previously, PCR-selected suppression subtraction hybridization analysis was executed to identify unique molecules functioning in the equine endometrium during periods of pregnancy establishment, and granzyme B (GZMB) cDNA was found in the pregnant endometrial cDNA library. Because GZMB is produced from natural killer (NK) cells, endometrial expression of GZMB and immune-related transcripts were characterized in ...
Sarcoids are the mostcommon skin tumors in horses, characterized by rare regression, invasiveness and high recurrence following surgical intervention and Delta Papillomaviruses are widely recognized as the causative agents of the disease. In order to gain new insights into equine sarcoid development, we have evaluated, in 25 equine sarcoids, by immunohistochemistry and western blotting analysis, the expression levels of VEGF, Ki67 and bcl-2. Moreover, we have measured microvessel density and specific vessel parameters. Results: All sarcoid samples showed a strong and finely granular cytoplasma...
Jung Y, Yoon M.Endocrine, paracrine, and autocrine factors orchestrate the development and physiology of the stallion reproductive system. Oxytocin (OXT) is one of the critical endocrine, paracrine, and autocrine factors for the male reproductive system. Previous studies have investigated OXT receptor (OXTR) expression in testes and epididymides, including humans, marmosets, macaques, swine, and sheep. This study aimed to explore (1) OXTR localization in the testes and epididymides and (2) the seasonal modification of OXTR expression in the testes. Adult stallion testis and epididymis samples were prepared u...
Miller MM, Sweeney CR, Russell GE, Sheetz RM, Morrow JK.To determine effects of blood contamination on western blot (WB) analysis of CSF samples for detection of anti-Sarcocystis neurona antibodies, and on CSF albumin and IgG concentrations, albumin quotient (AQ), and IgG index in horses. Methods: Prospective in vitro study. Methods: Blood with various degrees of immunoreactivity against S neurona was collected from 12 healthy horses. Cerebrospinal fluid without immunoreactivity against S neurona was harvested from 4 recently euthanatized horses. Methods: Blood was serially diluted with pooled nonimmunoreactive CSF so that final dilutions correspon...
Zhang L, Lei Y, Liu X, Wang X, Liu Z, Li D, Zheng P, Zhang L, Chen S, Xie P.Borna disease virus (BDV) is a neurotropic, enveloped, non-segmented, negative-stranded RNA virus that infects a wide variety of vertebrate species from birds to humans across a broad global geographic distribution. Animal symptomatology range from asymptomatic infection to behavioral abnormalities to acute meningoencephalitis. Asymptomatic BDV infection has been shown to be more frequent than conventionally estimated. However, the molecular mechanism(s) underyling asymptomatic BDV infection remain largely unknown. Here, based on real-time quantitative PCR and Western blotting, a total of 18 h...
Pittaway CE, Lawson AL, Coles GC, Wilson AD.Infection of horses with Anoplocephala perfoliata induces a severe inflammatory reaction of the caecal mucosa around the site of parasite attachment adjacent to the ileocecal valve. Lesions show epithelial erosion or ulceration of the mucosa with infiltration by eosinophils, lymphocytes and mast cells leading to oedema, gross thickening and fibrosis of the caecal wall. Despite this evidence of an inflammatory reaction to A. perfoliata within the mucosa of the caecum there is little information about the nature of the local immune response to A. perfoliata. An ELISA which assays serum IgG(T) an...
Duehlmeier R, Hacker A, Widdel-Bigdely A, von Engelhardt W, Sallmann HP.Glucose homeostasis depends on insulin-regulated glucose uptake in the skeletal muscles and fat tissues via glucose transporter (GLUT) 4 translocation into cellular plasma membranes. The present study sought to elucidate GLUT4 expression, GLUT1 and GLUT4 translocation and glucose uptake in the skeletal muscles of Shetland ponies. Semitendinosus muscle explants were removed by open muscle biopsy from six Shetland pony geldings under general anaesthesia. The expression of GLUT4 was analysed by measuring muscle crude membrane (CM) GLUT4 protein contents. To determine the insulin-stimulated GLUT t...
Młodawska W, Grzesiak M, Kochan J, Nowak A.Steroidogenic activity in the equine ovary from birth to puberty has been poorly investigated. This study aimed to examine the capability of the ovarian follicles of prepubertal and pubertal fillies to produce steroid hormones and to evaluate the expression and cellular localization of androgen receptor (AR) in their ovaries. The ovaries of 6-18 month-old fillies were divided into two groups: prepubertal (PrP) - without preovulatory follicle (pF) and corpus luteum (CL), and ovulating/postpubertal (Ov/pB) - with pF and/or CL in at least one of the gonads. Adult mares (Me) were used as a control...
Albrizio M, Lacalandra GM, Micera E, Guaricci AC, Nicassio M, Zarrilli A.Opioid receptors and endogenous opioid peptides act not only in the control of nociceptive pathways, indeed several reports demonstrate the effects of opiates on sperm cell motility and morphology suggesting the importance of these receptors in the modulation of reproduction in mammals. In this study we investigated the expression of delta opioid receptors on equine spermatozoa by western blot/indirect immunofluorescence and its relationship with sperm cell physiology. Methods: We analyzed viability, motility, capacitation, acrosome reaction and mitochondrial activity in the presence of naltri...
Kornicka K, Al Naem M, Röcken M, Zmiertka M, Marycz K.Osteochondritis dissecans (OCD) in equids, especially in sport horses, has become a growing issue as it contributes to the occurrence of lameness. Thus the aim of this study was to investigate the cytophysiological properties of OCD chondrocytes including expression of chondrogenic genes, apoptosis, mitochondria dynamics and autophagy. Horse chondrocytes were isolated from healthy (HE) and OCD cartilages. Properties of cells were evaluated using multiple assays e.g., polymerase chain reaction (PCR), immunofluorescence, Western blot. OCD chondrocytes were characterized by increased apoptosis an...
Sheehy JG, Hellyer PW, Sammonds GE, Mama KR, Powers BE, Hendrickson DA, Magnusson KR.To evaluate mu-opioid receptors in synovial membranes of horses and determine whether these receptors are up-regulated in nerve endings during inflammation. Methods: Synovial tissue obtained from 39 client-owned horses during arthroscopy and 14 research horses during necropsy; brain and synovial tissues were obtained during necropsy from 1 horse, and control tissues were obtained from a mouse. Methods: Horses were classified into 7 groups on the basis of histologically determined degree of inflammation. Binding of primary rabbit antibody developed against mu-opioid receptors in equine synovial...
Albrizio M, Guaricci AC, Maritato F, Sciorsci RL, Mari G, Calamita G, Lacalandra GM, Aiudi GG, Minoia R, Dell'Aquila ME, Minoia P.The development of fertilizing ability in sperm cells is associated with changes in the plasma membrane. However, to date the exact nature of sequentially activated primary receptors and channels and the signal transduction pathways derived from these remains elusive. We analyzed the expression and localization of the mu-opioid receptor in equine spermatozoa. A transcript corresponding to the third extracellular loop that selectively binds mu agonists was amplified, sequenced and compared with the known sequences in humans, rats and cattle. The amplification product showed a high degree of nuc...
Sugiura T, Nakamura H.The zinc content in platelets from rabbits, humans and horses was determined, and the levels of zinc were found to be significantly higher (3 micrograms/10(10) cells) than those in other peripheral blood cells. About 70% of the zinc in the supernatants of platelet lysates could be detected. From the results of gel filtration analysis, the zinc in platelet lysates was found to be bound with a low-molecular-weight protein (MW 6,000-8,000) detected as metallothionein (MT) on the basis of antigenic properties determined by enzyme-linked immunoassay and immunoblotting analysis using monoclonal anti...
Fortin JS, Benskey MJ, Lookingland KJ, Patterson JS, Howey EB, Goudreau JL, Schott HC.Pituitary pars intermedia dysfunction (PPID) develops slowly in aged horses as degeneration of hypothalamic dopaminergic neurons leads to proliferation of pars intermedia (PI) melanotropes through hyperplasia and adenoma formation. Dopamine (DA) concentrations and tyrosine hydroxylase (TH) immunoreactivity are markedly reduced in PI tissue of PPID-affected equids and treatment with the DA receptor agonist pergolide results in notable clinical improvement. Thus, we hypothesized that pergolide treatment of PPID-affected horses would result in greater DA and TH levels in PI tissue collected from ...
Szóstek AZ, Galvão AM, Ferreira-Dias GM, Skarzynski DJ.This study aimed to evaluate the influence of ovarian steroids on equine endometrial epithelial and stromal cells, specifically i) prostaglandin (PG) production in a time-dependent manner, ii) specific PG synthases mRNA transcription and protein expression, and iii) cell proliferation. After passage I, cells were exposed to vehicle, oxytocin (OT, positive control, 10(-7) M), progesterone (P4, 10(-7) M), 17β estradiol (E2, 10(-9) M), or P4+E2 for 12, 24, 48, or 72 h. Following treatment, PG concentration was determined using the direct enzyme immunoassay (EIA) method. Alterations in PG synth...
Sofronic-Milosavljevic Lj, Ilic N, Djordjevic M, Savic M, Gruden-Movsesijan A, Cuperlovic K, Murrell KD.In the Balkan countries, where trichinellosis is a re-emerging zoonosis, it is of great importance to determine Trichinella infection prevalence among the major hosts, including horses. One method for monitoring prevalence is serological surveillance; however, the validity of serological methods in horses is not well understood. The dynamics of anti-Trichinella IgG production and circulating excretory/secretory (ES) antigens were investigated in three horses experimentally-infected with Trichinella spiralis. Horses were slaughtered at 32 week post infection (p.i.). Low worm burdens were found ...
Galan JE, Timoney JF.Purpura hemorrhagica is an acute disease of the horse characterized by edema of the head and limbs, leucocytoclastic vasculitis, petechial hemorrhages in mucosae, musculature and viscera, and sometimes glomerulonephritis. It is usually associated with strangles, an upper respiratory tract disease of the horse caused by Streptococcus equi. We have detected and characterized immune complexes in the sera of horses with poststrangles purpura hemorrhagica by using PEG precipitation and Western blot analysis. The immune complexes contained IgA and S. equi-specific antigens similar to those found in ...
Alvarez I, Gutierrez G, Ostlund E, Barrandeguy M, Trono K.We analyzed the performance of a single-band Western blot (WB) test using recombinant p26 (rp26) capsid protein of equine infectious anemia virus. According to the results obtained, the rp26 WB test is a reliable confirmatory diagnostic tool to be used as a complementary test after an enzyme-linked immunosorbent assay or agar gel immunodiffusion test yielding doubtful results.
Malecki TM, Jillson GP, Thilsted JP, Elrod J, Torrez-Martinez N, Hjelle B.To determine whether animals had serologic evidence of infection with Sin Nombre virus (SNV). Methods: Prospective serosurvey. Methods: Serum samples were obtained from 145 cats, 85 dogs, 120 horses, and 24 cattle between April 1993 and August 1994 and 54 coyotes between December 1994 and February 1995. Methods: Serum samples were analyzed by western immunoblot assays for reaction with SNV nucleocapsid antigen. Samples with reactivity to SNV nucleocapsid proteins were used to probe multiple-antigen blots containing recombinant fusion proteins derived from prototypic hantaviruses. Lung tissue o...
Schnabel CL, Wagner S, Wagner B, Durán MC, Babasyan S, Nolte I, Pfarrer C, Feige K, Murua Escobar H, Cavalleri JM.Research on equine cytokines is often performed by analyses of mRNA. For many equine cytokines an analysis on the actual protein level is limited by the availability of antibodies against the targeted cytokines. Generation of new antibodies is ongoing but time consuming. Thus, testing the reactivity of commercially available antibodies for cross-reactivity with equine cytokines is of particular interest. Fifteen monoclonal antibodies against IL-1β, IL-6, IL-8, IL-12, IL-18 and Granulocyte Macrophage Colony stimulating factor (GM-CSF) of different species were evaluated for reactivity with the...
King SS, Campbell AG, Dille EA, Roser JF, Murphy LL, Jones KL.Dopamine (DA) agonist and antagonist treatments can affect ovarian reproductive events in the mare. To support our theory that DA produces these effects by acting directly on the ovary, we analyzed equine ovarian tissues for the presence of dopamine receptor-1 (D1r) and dopamine receptor-2 (D2r) mRNA by reverse transcription polymerase chain reaction (RT-PCR) and D1r and D2r proteins by Western blot and immunohistochemistry (IHC). RT-PCR was performed on RNA isolated from ovarian cortex, medulla, granulosa/theca or corpus luteum (CL) tissues and from pituitary (D2r control) and renal artery (D...
