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.
Rapacz-Leonard A, Leonard M, Chmielewska-Krzesińska M, Paździor-Czapula K, Janowski T.Major histocompatibility protein class I (MHC-I) is believed to be expressed in the horse allantochorion only in limited areas at limited times. However, its expression has only been investigated in early pregnancy with non-quantitative techniques that cannot reliably detect small amounts of protein. To quantify the relative expression of MHC-I in the allantochorion and endometrium during days 90-240 of pregnancy (PREG), parturition with physiological delivery of fetal membranes (PHYS), and parturition with retention of these membranes (FMR). Also, to visualize protein expression and determine...
Bonometti S, Menarim BC, Reinholt BM, Ealy AD, Johnson SE. To provide insight into maternal recognition of pregnancy control in equids, the mitogenic and developmental effects of endometrium-expressed growth factors (epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1)) were examined in equine iTr cells, an equine trophectoderm cell line. Initial western blots revealed that HGF and IGF-1 stimulate phosphorylation of AKT serine/threonine kinase 1 (AKT1) and EGF, FGF2, or HGF resulted in phosphorylation of both extracellular signal-regulated kinase 1 (ERK1) and ERK2...
Boere J, van de Lest CHA, de Grauw JC, Plomp SGM, Libregts SFWM, Arkesteijn GJA, Malda J, Wauben MHM, van Weeren PR.Extracellular vesicle (EV) concentration, characteristics and function in equine synovial fluid (SF) during normal growth and development has not previously been studied. Isolation of EVs was performed in SF from three healthy foals and two adult horses by differential ultracentrifugation (10,000g and 200,000g); EVs were purified by sucrose density gradient floatation and analysed by high-resolution flow cytometry (FCM), buoyant density and western blotting. Additionally, repeated biomarker analysis of sulphated glycosaminoglycans (GAG), matrix metalloproteinase (MMP), C-terminal crosslinked t...
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...
Ortega-Ferrusola C, Martin Muñoz P, Ortiz-Rodriguez JM, Anel-López L, Balao da Silva C, Álvarez M, de Paz P, Tapia JA, Anel L, Silva-Rodríguez A....We hypothesized that thiols and particularly glutathione (GSH) are essential for the regulation of stallion sperm functionality. To test this hypothesis, we initially investigated the relationship between sperm function and GSH content, revealing highly significant correlations between GSH, sperm viability, motility, and velocity parameters (P < 0.001). Furthermore, the deleterious effects of GSH depletion using menadione and 1,3 dimethoxy 1,4, naphtoquinone (DMNQ) were able to be prevented by the addition of cysteine, but no other antioxidant. Pre-incubation with cysteine prevented menad...
Rivera Del Alamo MM, Reilas T, Galvão A, Yeste M, Katila T.Treatment with intrauterine devices (IUD) prolongs luteal phases in mares, but the mechanism for this has not been fully elucidated. The aims of the present study were to examine how IUDs affect the uterus to induce longer luteal phases, particularly the role of cyclooxygenase-2 (COX-2) in the maintenance of the corpus luteum (CL). Twenty-seven reproductively normal mares were included: 12 were inseminated (AI), and 15 were fitted with IUDs. Blood samples for progesterone were obtained on Days 0, 3, 5, 7, 9, 11, 13, 14, and 15 (relative to day of ovulation). The groups were further divided int...
Furusawa Y, Yamada S, Itai S, Sano M, Nakamura T, Yanaka M, Handa S, Mizuno T, Maeda K, Fukui M, Harada H, Kaneko MK, Kato Y.Podoplanin (PDPN), a type I transmembrane glycoprotein, is expressed in several body tissues, including podocytes of renal glomerulus, type I alveolar cells of lung, and lymphatic endothelial cells. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) presented on platelets. Monoclonal antibodies (mAbs) against human-, mouse-, rat-, rabbit-, dog-, bovine-, and cat-PDPN have already been established. However, anti-horse PDPN mAbs have not yet been developed. In this study, we immunized mice with synthetic horse PDPN peptides and developed anti-horse PDPN mAbs...
Szóstek-Mioduchowska AZ, Lukasik K, Skarzynski DJ, Okuda K.Transforming growth factor (TGF)-β1 not only regulates cell growth, development, and tissue remodeling, but it also participates in the pathogenesis of tissue fibrosis. In the equine endometrium, the concentration of TGF-β1 is correlated with endometrosis (equine endometrial fibrosis). In other tissues, TGF-β1 induces differentiation of many cell types into myofibroblasts. These cells are characterized by α-smooth muscle actin (α-SMA) expression and an ability to deposit excessive amounts of extracellular matrix (ECM) components. The aim of the study was to determine whether TGF-β1 plays...
Read JE, Cabrera-Sharp V, Offord V, Mirczuk SM, Allen SP, Fowkes RC, de Mestre AM.Equine chorionic girdle trophoblast cells play important endocrine and immune functions critical in supporting pregnancy. Very little is known about the genes and pathways that regulate chorionic girdle trophoblast development. Our aim was to identify genes and signalling pathways active in vivo in equine chorionic girdle trophoblast within a critical 7-days window. We exploited the late implantation of the equine conceptus to obtain trophoblast tissue. An Agilent equine 44K microarray was performed using RNA extracted from chorionic girdle and chorion (control) from equine pregnancy days 27, ...
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...
Mendoza E, Bubis J, Pérez-Rojas Y, Montilla AJ, Spencer LM, Bustamante F, Martínez JC.Previously, we have identified a protein in Trypanosoma equiperdum that possesses homology with the regulatory (R) subunits of the mammalian cAMP-dependent protein kinase (PKA). The recombinant T. equiperdum PKA R-like protein was expressed in bacteria and purified to homogeneity. Mice polyclonal antibodies were raised against the recombinant R-like protein to serologically evaluate its humoral immune response. High titers of specific sera antibodies were obtained against the parasite R-like protein by indirect enzyme-linked immunosorbent assay (ELISA), and immunoblots revealed that this prote...
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...
Sifi A, Adi-Bessalem S, Laraba-Djebari F.Antivenom treatment has been largely used against scorpion stings. Despite their efficacy, the use of mammalian antivenoms may cause adverse effects due to the immune system activation. IgYs from hyperimmunized laying hens against venoms could be a promising alternative to equine IgGs due to the various benefits that these antibodies can provide. Here we report the preparation of specific IgYs by immunizing laying hens with Aah (Androctonus australis hector) scorpion venom. IgYs were isolated from egg yolks by water dilution and salt precipitation methods; they were characterized by sodium dod...
Read JE, Cabrera-Sharp V, Kitscha P, Cartwright JE, King PJ, Fowkes RC, de Mestre AM.Equine chorionic gonadotrophin (eCG) is a placental glycoprotein critical for early equine pregnancy and used therapeutically in a number of species to support reproductive activity. The factors in trophoblast that transcriptionally regulate eCGβ-subunit (), the gene which confers the hormones specificity for the receptor, are not known. The aim of this study was to determine if glial cells missing 1 regulates promoter activity. Here, studies of the proximal promoter identified four binding sites for glial cells missing 1 (GCM1) and western blot analysis confirmed GCM1 was expressed in equi...
Jiménez-Trejo F, Coronado-Mares I, Boeta M, González-Santoyo I, Vigueras-Villaseñor R, Arriaga-Canon C, Herrera LA, Tapia-Rodríguez M.Assisted reproductive techniques have been used on several domestic animals to preserve desirable traits in strains of high genetic and commercial value; however in equines its efficiency rate is relatively low. To increase the conception ratio in stallions, some research groups have used pharmacological treatments which promote sperm hyperactivation in order to increase male's fertility rates. In this way, our previous work suggests that serotonin (5-HT) could be a good pharmacological candidate that facilitates conception rate in domestic horses. 5-HT is a neurohormone involved in several re...
Ceriotti S, Consiglio AL, Casati L, Cremonesi F, Sibilia V, Ferrucci F.Increasing evidence suggests a role for ghrelin in the control of articular inflammatory diseases like osteoarthritis (OA). In the present study we examined the ability of ghrelin to counteract LPS-induced necrosis and apoptosis of chondrocytes and the involvement of GH secretagogue receptor (GHS-R)1a in the protective action of ghrelin. The effects of ghrelin (10-10 mol/L) on equine primary cultured chondrocytes viability and necrosis in basal conditions and under LPS treatment (100 ng/ml) were detected by using both acridine orange/propidium iodide staining and annexin-5/propidium iodide...
D'Ascola A, Bruschetta G, Zanghì G, Campo S, Medica P, Campana S, Ferlazzo G, Gibbs BF, Ferlazzo AM.Serotonin (5-HT) is a neurohormone transported from plasma into platelets and leukocytes by a specific transporter (SERT). While it is known that the brain 5-HT system is modulated by physical exercise, the peripheral serotoninergic response to exercise is not yet fully elucidated. In particular, this study aimed to evaluate changes in plasma 5-HT levels and equine leukocyte SERT expression in response to treadmill exercise in untrained horses. Analyses were carried out pre- and post-treadmill exercise. 5-HT plasma levels were analysed by HPLC. Leukocytes and platelets were isolated to perform...
Qi T, Hu Y, Hu Z, Zhao S, Cullinane A, Lyons P, Gildea S, Wang X.Quantitation of virions is one of the important indexes in virological studies. To establish a sensitive and rapid quantitative detection method for equine arteritis virus (EAV), an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed by using two EAV nucleoprotein monoclonal antibodies (mAbs), 2B9 and 2B3, prepared in this study. After condition optimization, mAb 2B9 was used as the capture antibody, and HRP-labeled 2B3 was chosen as the detecting antibody. The AC-ELISA had a good standard curve when viral particles of the Bucyrus EAV strain were used as a reference stan...
Dal Ben V, Oliveira RS, Borchardt JL, Valente JSS, Brasil CL, Zambrano CG, Leite FPL, Botton SA, Pereira DIB.Pythium insidiosum is an important aquatic Oomycota that causes pythiosis in mammals, especially horses, dogs, and humans; these inhabit marshy environments in tropical and subtropical areas. The aim of this study was to determine the protein profile, as well as identify likely immunodominant proteins, of Brazilian P. insidiosum isolates from southern Brazil, an important equine pythiosis endemic area. P. insidiosum isolates (horses, n = 20 and dogs, n = 02) were analyzed by SDS-PAGE and Western blot techniques. Horse, cattle, dog, and rabbit sera of both diseased and healthy animals were used...
Hayashi S, Tanaka T, Moriishi K, Hirayama K, Yamada A, Hotta K.Non-primate hepacivirus (NPHV) is recently identified as a closely related homologue of hepatitis C virus. The previous studies showed a high prevalence of NPHV infection among Japanese domestic horses originated from abroad. The historical distribution of NPHV among horses in Japan, therefore, is still unknown. In this study, seroepidemiological study of NPHV was conducted using 335 sera from five breeds of Japanese native horses. These horses are maintained as the pedigree and are reared apart from other horse breeds. The detection of antibodies against NPHV were conducted by western blot an...
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...
Freer H, Hillegas JM, Wimer C, Baldwin C, LaBresh J, Wagner B.Interleukin-2 (IL-2) is a T cell growth factor and major modulator of T helper (Th) cell differentiation. Here, we have developed and characterized a monoclonal antibody to equine IL-2 (anti-IL-2 mAb, clone 158-1). The IL-2 mAb detected rIL-2 by ELISA, intracellular staining and flow cytometry analysis and Western blotting. The IL-2 mAb was also paired with a polyclonal IL-2 detection antibody in both ELISA and a fluorescent bead-based assay. When these two assays were compared using identical reagents there was an improved analytical sensitivity (46pg/ml) and wider linear quantification range...
Ghori MT, Khan MS, Khan JA, Rabbani M, Shabbir MZ, Chaudhry HR, Ali MA, Muhammad J, Elschner MC, Jayarao BM.Glanders is an infectious and contagious bacterial disease of equines. A little is known about its seroprevalence and risk factors in working equines in countries where the disease is endemic. Also, there are no reports on prevalence of the disease in areas where there is a prior evidence of Burkholderia (B.) mallei detection in soil. A cross-sectional study was conducted in selected districts (n=09) of Punjab province of Pakistan during 2014-2015. A total of 1008 serum samples were screened for detection of antibodies to B. mallei with complement fixation test followed by western blot. The ov...
Abo-Aziza FAM, Hendawy SHM, Namaky AHE, Ashry HM.The aim of this study was to investigate the early diagnosis of strongyle infection based on early changes in Th1 and Th2 cytokines beside the diagnostic accuracy values and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting profiles using prepared strongyles antigens. Methods: A total of 73 donkeys had a mean age of 4-32 years old were parasitologically examined for strongyle infection. The early changes in Th1 and Th2 cytokines were determined, and the diagnostic accuracy values and SDS-PAGE and western blotting profiles were performed using prepared st...
Warnken T, Brehm R, Feige K, Huber K.The aim of the study was to analyze key proteins of the equine insulin signaling cascade and their extent of phosphorylation in biopsies from muscle tissue (MT), liver tissue (LT), and nuchal AT, subcutaneous AT, and retroperitoneal adipose tissues. This was investigated under unstimulated (B1) and intravenously insulin stimulated (B2) conditions, which were achieved by injection of insulin (0.1 IU/kg bodyweight) and glucose (150 mg/kg bodyweight). Twelve warmblood horses aged 15 ± 6.8 yr (yr), weighing 559 ± 79 kg, and with a mean body condition score of 4.7 ± 1.5 were included in the s...
Gutiérrez-Expósito D, García-Bocanegra I, Howe DK, Arenas-Montes A, Yeargan MR, Ness SL, Ortega-Mora LM, Álvarez-García G.Equine besnoitiosis, caused by Besnoitia bennetti, and equine protozoal myeloencephalitis (EPM), caused by Sarcocystis neurona and Neospora hughesi are relevant equine diseases in the Americas that have been scarcely studied in Europe. Thus, a serosurvey of these cystogenic coccidia was carried out in Southern Spain. A cross-sectional study was performed and serum samples from horses (n = 553), donkeys (n = 85) and mules (n = 83) were included. An in-house enzyme-linked immunosorbent assay (ELISA) was employed to identify a Besnoitia spp. infection and positive results were confirmed by ...
Braus BK, Miller I, Kummer S, Kleinwort KJH, Hirmer S, Hauck SM, McMullen RJ, Kerschbaumer M, Deeg CA.Immune mediated keratitis (IMMK) is primarily a non-ulcerative keratitis in horses causing intermittent ocular pain, eventually resulting in visual impairment. Affected horses typically respond to immunomodulatory treatment. However, the underlying cause of the disease remains enigmatic. The current study was undertaken to investigate the presence of autoantibodies in horses with immune mediated keratitis. Using 28 horses with IMMK and 27 healthy controls screening for serum autoantibodies against the corneal proteome using indirect immunofluorescence, one-dimensional (1DE) and two-dimensional...
Lane HE, Burns TA, Hegedus OC, Watts MR, Weber PS, Woltman KA, Geor RJ, McCutcheon LJ, Eades SC, Mathes LE, Belknap JK.Insulin dysregulation, obesity, and exposure to high-nonstructural carbohydrate (NSC) forage are risk factors for equine metabolic syndrome-associated laminitis (EMSAL); high systemic insulin concentrations in EMSAL are proposed to induce cellular dysregulation in the digital lamellae through activation of the insulin-like growth factor-1 receptor. Objective: To use a dietary challenge model (DCM) and a euglycaemic-hyperinsulinaemic clamp (EHC) model to assess lamellar growth factor-related signalling. Methods: Lamellar phospho (P)-protein concentrations of signalling proteins important in gro...
Nascimento DA, Vieira RF, Vieira TS, Toledo RD, Tamekuni K, Santos NJ, Gonçalves DD, Vieira ML, Biondo AW, Vidotto O.The aims of the present study were to serosurvey dogs, horses, and humans highly exposed to tick bites for anti-Borrelia burgdorferi s.l. antibodies, identify tick species present, and determine risk factors associated with seropositivity in a rural settlement of Paraná State, southern Brazil. Eighty-seven residents were sampled, along with their 83 dogs and 18 horses, and individual questionnaires were administered. Immunofluorescence antibody test (IFAT) was performed on serum samples and positive samples were subjected to western blot (WB) analysis. Anti-B. burgdorferi antibodies were foun...
Lee G, Jung H, Yoon M.The molecular markers for specific germ cell stages can be utilized for identifying, monitoring, and separating a particular stage of germ cells. The RNA-binding protein Lin28 is expressed in gonocytes of human fetal testes. The Lin28 expression is restricted to a very small population of spermatogonial cells in human, mice, and monkey. The main objective of this study was to investigate the expression pattern of Lin28 in stallion testes at different reproductive stages. Based on the presence or absence of full spermatogenesis and lumina in seminiferous tubules, the testicular samples were cat...
Baumber J, Sabeur K, Vo A, Ball BA.The objective of this study was to examine the influence of reactive oxygen species (ROS) on equine sperm capacitation. Motile equine spermatozoa were separated on a discontinuous Percoll gradient, resuspended at 10 x 10(6)ml in Tyrode's medium supplemented with BSA (0.5%) and polyvinyl alcohol (0.5%) and incubated at 39 degrees C for 2h with or without the xanthine (X; 0.1mM)-xanthine oxidase (XO; 0.01 U/ml) system or NADPH (0.25 mM). The importance of hydrogen peroxide or superoxide for capacitation was determined by the addition of catalase (CAT; 150 U/ml) or superoxide dismutase (SOD; 150 ...
Waller AP, Kohler K, Burns TA, Mudge MC, Belknap JK, Lacombe VA.Although the importance of adipose tissue (AT) glucose transport in regulating whole-body insulin sensitivity is becoming increasingly evident and insulin resistance (IR) has been widely recognized, the underlying mechanisms of IR are still not well understood. The purpose of the present study was to determine the early pathological changes in glucose transport by characterizing the alterations in glucose transporters (GLUT) in multiple visceral and subcutaneous adipose depots in a large animal model of naturally occurring compensated IR. AT biopsies were collected from horses, which were clas...
Madigan JE, Rikihisa Y, Palmer JE, DeRock E, Mott J.The original objective was to determine seroprevalence of Ehrlichia risticii antibody among horses in California. On the basis of the unexpected results of the survey, an investigation into the accuracy and reproducibility of results of the indirect fluorescent antibody (IFA) test for E risticii was carried out. Methods: Prospective, seroprevalence study. Methods: Healthy horses (n = 655) and horses with clinical signs of equine monocytic ehrlichiosis (EME; n = 514) from various regions of California. Methods: The IFA test was performed. Results were compared with results of an ELISA and with ...
Fenger CK, Granstrom DE, Gajadhar AA, Williams NM, McCrillis SA, Stamper S, Langemeier JL, Dubey JP.Sarcocystis sp. sporocysts isolated from eight feral opossums (Didelphis virginiana) were pooled and fed to 18 commercially reared budgerigars (Melopsittacus undulatus), 14 wild-caught sparrows (Passer domesticus), one wild-caught slate-colored Junco (Junco hyemalis) and five weanling horses (Equus caballus). All budgerigars died within 5 weeks post inoculation (wpi). Histologic examination revealed meronts within the pulmonary epithelia and typical Sarcocystis falcatula sarcocysts developing in the leg muscles. Sparrows were euthanized 13 and 17 wpi and their carcasses were fed to four labora...
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 ...
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...
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...
Wilson AD, Heesom KJ, Mawby WJ, Mellor PS, Russell CL.IgE-mediated type 1 hypersensitivity reactions to the bites of insects are a common cause of skin disease in horses. Insect bite hypersensitivity (IBH) is most frequently associated with bites of Culicoides spp. and occurs in all parts of the world where horses and Culicoides coexist. The main allergens that cause IBH are probably some of the abundant proteins in the saliva of Culicoides associated with blood feeding. Western blots of Culicoides proteins separated by 1D gel-electrophoresis detected strong IgE responses in all horses with IBH to antigens in protein extracts from wild caught Cul...
Schulze-Tanzil G, Müller RD, Kohl B, Schneider N, Ertel W, Ipaktchi K, Hünigen H, Gemeinhardt O, Stark R, John T.For lack of sufficient human cartilage donors, chondrocytes isolated from various animal species are used for cartilage tissue engineering. The present study was undertaken to compare key features of cultured large animal and human articular chondrocytes of the knee joint. Primary chondrocytes were isolated from human, porcine, ovine and equine full thickness knee joint cartilage and investigated flow cytometrically for their proliferation rate. Synthesis of extracellular matrix proteins collagen type II, cartilage proteoglycans, collagen type I, fibronectin and cytoskeletal organization were ...
Phillips T, Ferraz I, Bell S, Clegg PD, Carter SD, Mobasheri A.Glucose serves as the major energy substrate for articular chondrocytes and as the main precursor for the synthesis of extracellular matrix glycosaminoglycans in cartilage. Chondrocytes have been shown to express several glucose transporter (GLUT) isoforms including GLUT1 and GLUT3. The aim of this investigation was to determine the effects of endocrine and cytokine factors on the capacity of equine articular chondrocytes for transporting 2-deoxy-d-[2,6-3H] glucose and on the expression levels of GLUT1 and GLUT3. Chondrocytes maintained in monolayer culture were stimulated for 24 h with TNF-al...
Nagata LP, Hu WG, Masri SA, Rayner GA, Schmaltz FL, Das D, Wu J, Long MC, Chan C, Proll D, Jager S, Jebailey L, Suresh MR, Wong JP.The efficacy of a DNA vaccine against western equine encephalitis (WEE) infection in mice was evaluated. The 26S structural region was expressed, in vitro from an internal T7 promoter using a rabbit reticulysate transcription/translation system; and from a CMV promoter after transfection into Vero cell monolayers. The proteins synthesized were reactive with anti-WEE virus (WEEV) antibodies, both in western blot analysis and histochemical staining, respectively. When the DNA vaccine plasmid, pVHX-6, was administered intraepidermally to mice, followed by challenge in a lethal mouse model, the le...
Deeg CA, Eberhardt C, Hofmaier F, Amann B, Hauck SM.Autoimmune uveitis is an intraocular inflammation that arises through autoreactive T-cells attacking the inner eye, eventually leading to blindness. However, the contributing molecular pathomechanisms within the affected tissues remain as yet elusive. The extracellular matrix (ECM) is a highly dynamic structure that varies tremendously and influences the encompassing tissue. In order to assess ECM re-modeling in autoimmune uveitis, we investigated the expression of ECM molecules fibronectin and osteopontin in vitreous and retina samples. This was carried out in the only spontaneous animal mode...
D'Ascola A, Bruschetta G, Zanghì G, Campo S, Medica P, Campana S, Ferlazzo G, Gibbs BF, Ferlazzo AM.Serotonin (5-HT) is a neurohormone transported from plasma into platelets and leukocytes by a specific transporter (SERT). While it is known that the brain 5-HT system is modulated by physical exercise, the peripheral serotoninergic response to exercise is not yet fully elucidated. In particular, this study aimed to evaluate changes in plasma 5-HT levels and equine leukocyte SERT expression in response to treadmill exercise in untrained horses. Analyses were carried out pre- and post-treadmill exercise. 5-HT plasma levels were analysed by HPLC. Leukocytes and platelets were isolated to perform...
Harty RN, Caughman GB, Holden VR, O'Callaghan DJ.Equine herpesvirus 1 (EHV-1, Kentucky A strain) preparations enriched for defective interfering particles (DIPs) can readily establish persistent infection. The UL1 gene, which is conserved in the genome of DIPs that mediate persistent infection, maps between nucleotides 1418 and 2192 (258 amino acids) from the L (long) terminus. UL1 has no homology with any known gene encoded by herpes simplex virus type 1 but has limited homology to open reading frame 2 of varicella-zoster virus and the "circ" gene of bovine herpesvirus type 1. Previous work showed that the EHV-1 UL1 gene belongs to the earl...
Furr M, Kennedy T, MacKay R, Reed S, Andrews F, Bernard B, Bain F, Byars D.Equine protozoal myeloencephalitis (EPM) is a neurologic disease of horses most commonly caused by the protozoan parasite Sarcocystis neurona. Until recently the only treatment option was the combination of a sulfonamide with pyrimethamine. The present study was performed to assess the efficacy of ponazuril, an anticoccidial triazine-based compound, as a treatment for naturally occurring EPM. One hundred one horses with EPM were randomly allocated to treatment with ponazuril 15% oral paste at either 5 or 10 mg/kg body weight for 28 consecutive days. Horses were evaluated clinically and by anal...
Wooldridge AA, Edwards HG, Plaisance EP, Applegate R, Taylor DR, Taintor J, Zhong Q, Judd RL.To characterize adiponectin protein complexes in lean and obese horses. Methods: 26 lean horses and 18 obese horses. Procedures-Body condition score (BCS) and serum insulin activity were measured for each horse. Denaturing and native western blot analyses were used to evaluate adiponectin complexes in serum. A human ELISA kit was validated and used to quantify high-molecular weight (HMW) complexes. Correlations between variables were made, and HMW values were compared between groups. Results: Adiponectin was present as a multimer consisting of HMW (> 720-kDa), low-molecular weight (180-kDa)...
Raulo SM, Sorsa T, Tervahartiala T, Pirilä E, Maisi P.Gelatinolytic activity was analysed to study whether elevated activity previously found at the tracheal level of the respiratory tract of horses with chronic obstructive pulmonary disease (COPD) could also be found at the lower part of the respiratory tract. Furthermore, presence and significance of the gelatinolytic matrix metalloproteinases (MMPs) MMP-2 and MMP-9 in respiratory secretions of healthy and COPD horses were determined. Elevated gelatinolytic matrix metalloproteinases were detected in bronchoalveolar and tracheobronchial secretions from COPD horses. The main pathologically elevat...
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...
Wilson AD, Harwood L, Torsteinsdottir S, Marti E.Immunoglobulin E forms a minor component of serum antibody in mammals. In tissues IgE is bound by FcvarepsilonRI receptors on the surface of mast cells and mediates their release of inflammatory substances in response to antigen. IgE and mast cells have a central role in immunity to parasites and the pathogenesis of allergic diseases in horses and other mammals. This paper describes the production of several novel monoclonal antibodies that detect native equine IgE in immunohistology, ELISA and Western blotting. An antigen capture ELISA to quantify equine IgE in serum has been developed using ...
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...
Proudman CJ, Trees AJ.Whole worm extract (WWE) and excretory/secretory (E/S) antigens of Anoplocephala perfoliata were characterised by SDS-PAGE and their use in the serodiagnosis of equine cestodosis was evaluated. An enzyme-linked immunosorbent assay (ELISA) was used to compare WWE and E/S antigen as the capture layer in an antibody capture ELISA. E/S antigen gave the best differentiation between sera from tapeworm-positive and tapeworm-negative horses. The E/S-ELISA was optimised and validated against sera from horses of known tapeworm status. This assay gave a diagnostic sensitivity of 68% (n = 38) and a specif...
Ghori MT, Khan MS, Khan JA, Rabbani M, Shabbir MZ, Chaudhry HR, Ali MA, Muhammad J, Elschner MC, Jayarao BM.Glanders is an infectious and contagious bacterial disease of equines. A little is known about its seroprevalence and risk factors in working equines in countries where the disease is endemic. Also, there are no reports on prevalence of the disease in areas where there is a prior evidence of Burkholderia (B.) mallei detection in soil. A cross-sectional study was conducted in selected districts (n=09) of Punjab province of Pakistan during 2014-2015. A total of 1008 serum samples were screened for detection of antibodies to B. mallei with complement fixation test followed by western blot. The ov...
Roberto da Costa RP, Ferreira-Dias G, Mateus L, Korzekwa A, Andronowska A, Platek R, Skarzynski DJ.Nitric oxide (NO) plays an important role in angiogenesis and in the regulation of the blood flow. This study was carried out to investigate (i) the effects of endogenous estrogens and progestins and exogenous progesterone (P(4)) (5 ng/ml or 1 microg/ml) or estradiol 17beta (E(2)beta) (50 pg/ml or 1 microg/ml) on in vitro endometrial NO synthesis; (ii) the presence of different isoforms of NO synthase; (iii) and their relationship to microvascular density in the equine endometrium during the estrous cycle. NOS expression was also evaluated in the myometrium. Expression of endothelial and induc...
Bosler EM, Cohen DP, Schulze TL, Olsen C, Bernard W, Lissman B.By using paired sera the IgM and IgG host responses were analyzed in dogs with ELISA and Western blot techniques. Antibodies in clinical seropositive dogs bound to 4-25 IgM and up to 40 or more IgG antigenic determinants. Early IgM response to the 41-kDa flagellin persisted for at least 9 months and involved as many as seven other peptides. IgG response expanded later in the disease and involved more immunogens than are currently recognized in late human disease. A percentage of asymptomatic dogs that later developed clinical symptoms were seropositive. Immunoblot studies suggested that B. bur...
Belin F, Goudet G, Duchamp G, Gérard N.The objective of the present study was to determine the changes in follicular fluid steroid concentrations and in granulosa cell steroidogenic enzyme expression during the follicular phase, in relation to follicular size and physiological status in the mare. Follicular fluid and follicular cells were recovered by ultrasound-guided follicular punctures either around the time of emergence of the dominant follicle, at the end of the dominant follicle growth, or at the preovulatory stage, after injection of gonadotropin to induce ovulation. Cellular relative amounts of steroidogenic acute regulato...
Hosaka Y, Kirisawa R, Ueda H, Yamaguchi M, Takehana K.Tumor necrosis factor (TNF) receptors (TNF-R)-mediated cell survival or apoptosis has been demonstrated in many cells, but little is known about survival or apoptotic signals via TNF-R1 in tendinocytes. In this study, we focused on four signaling factors, TNFalpha, TNF-R1, TNFR-associated factor2 (TRAF2) and caspase-3, in order to elucidate the signaling events in tendinocytes. Samples were obtained from normal, inflamed and scar-formed equine superficial digital flexor tendons. To detect these signaling factors, samples were subjected to immunohistochemistry and Western blot analysis, and som...
Hicks BA, Etter SJ, Carnahan KG, Joyce MM, Assiri AA, Carling SJ, Kodali K, Johnson GA, Hansen TR, Mirando MA, Woods GL, Vanderwall DK, Ott TL.Pregnancy and interferon-tau (IFN tau) upregulate uterine Mx gene expression in ewes; however, the only known role for Mx is in the immune response to viral infection. We hypothesize that Mx functions as a conceptus-induced component of the anti-luteolytic mechanism and/or regulator of endometrial secretion or uterine remodeling during early pregnancy. This study was conducted to determine the effects of early pregnancy on uterine Mx expression in domestic farm species with varied mechanisms of pregnancy recognition. Endometrium from cows, gilts, and mares was collected during the first 20 d o...
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...
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...
Amaral A, Fernandes C, Rebordão MR, Szóstek-Mioduchowska A, Lukasik K, Gawronska-Kozak B, Telo da Gama L, Skarzynski DJ, Ferreira-Dias G.Neutrophil extracellular traps (NETs) fight endometritis, and elastase (ELA), a protease found in NETs, might induce collagen type I (COL1) accumulation in equine endometrium. Metallopeptidases (MMPs) are involved in extracellular matrix balance. The aim was to evaluate the effects of ELA and sivelestat (selective elastase inhibitor) on MMP-2 and MMP-9 expression and gelatinolytic activity, as well as the potential inhibitory effect of sivelestat on ELA-induced COL1 in equine endometrium. Endometrial explants from follicular (FP) and mid-luteal (MLP) phases were treated for 24 or 48 h with ELA...
