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.
Burk SV, Dangoudoubiyam S, Brewster-Barnes T, Bryant UK, Howe DK, Carter CN, Vanzant ES, Harmon RJ, Kazacos KR, Rossano MG.Currently, diagnosis of Parascaris equorum infection in equids is limited to patent infections. The goals of this study were to culture P. equorum larvae in vitro and identify excretory-secretory (ES) products for prepatent diagnostic testing. Parascaris equorum L2/L3 larvae were hatched and cultured for up to 3 weeks for ES product collection. Fifth stage (L5) P. equorum were also cultured for ES product collection. Examination of ES fractions by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver stain revealed L2/L3 products ranging from 12-94 kDa and L5 products...
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...
Morrison PK, Bing C, Harris PA, Maltin CA, Grove-White D, Argo CM.Obesity, a major concern for equine welfare, is highly prevalent in the leisure horse population. Skeletal-muscle and adipose tissues are important determinants of maintenance energy requirements. The myostatin and perilipin pathways play key roles in the regulation of muscle mass and lipolysis respectively and have both been associated with obesity predisposition in other mammalian species. High quality samples, suitable for molecular biology, are an essential prerequisite for detailed investigations of gene and protein expression. Hence, this study has evaluated a) the post-mortem stability ...
Di Febo T, Luciani M, Ciarelli A, Bortone G, Di Pancrazio C, Rodomonti D, Teodori L, Tittarelli M.Monoclonal antibodies (MAbs) against horse IgG were produced by immunizing Balb/c mice with purified horse IgG and were characterized in indirect ELISA versus purified immunoglobulins from donkey, cow, buffalo, sheep, pig, and chicken. Three MAbs (1B10B6C9, 1B10B6C10, 1B10B6E9) reacted only with horse and donkey IgG and IgM and, in western blotting, were specific for the Fc fragment of equine IgG. MAb 1B10B6E9 was used in chemiluminescent immunoblotting assay for the diagnosis of dourine and in indirect immunofluorescence assay (IFA) for the diagnosis of African horse sickness and dourine.
da Silveira JC, Carnevale EM, Winger QA, Bouma GJ.Ovarian follicle growth and maturation requires extensive communication between follicular somatic cells and oocytes. Recently, intercellular cell communication was described involving cell-secreted vesicles called exosomes (50-150 nm), which contain miRNAs and protein, and have been identified in ovarian follicular fluid. The goal of this study was to identify a possible role of exosomes in follicle maturation. Methods: Follicle contents were collected from mares at mid-estrous (~35 mm, before induction of follicular maturation) and pre-ovulatory follicles (30-34 h after induction of folli...
Jung HJ, Song H, Yoon MJ.Deleted in azoospermia-like (DAZL) is used as a germ cell marker in several species, including mice, rats, pigs, rhesus monkeys, bulls, and humans. Our objectives with this study were to investigate DAZL expression in stallion germ cells by using immunofluorescence, immunocytochemistry, and western blotting, and to determine the effects of reproductive stage and breeding season on the DAZL-positive cell population in seminiferous tubule cross sections. Testes were obtained during routine castration procedures at a large animal clinic and routine field service castration. The reproductive stage...
Hdud IM, Mobasheri A, Loughna PT.The metabolic activity of articular chondrocytes is influenced by osmotic alterations that occur in articular cartilage secondary to mechanical load. The mechanisms that sense and transduce mechanical signals from cell swelling and initiate volume regulation are poorly understood. The purpose of this study was to investigate how the expression of two putative osmolyte channels [transient receptor potential vanilloid 4 (TRPV4) and large-conductance Ca(2+)-activated K(+) (BKCa)] in chondrocytes is modulated in different osmotic conditions and to examine a potential role for MAPKs in this process...
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...
Arkoun B, Gautier C, Delalande C, Barrier-Battut I, Guénon I, Goux D, Bouraïma-Lelong H.Among mammals, the stallion produces the largest amount of testicular estrogens. These steroid hormones are produced mainly by Leydig and Sertoli cells in the testis and also in the epididymis. Their role in horse testicular physiology and their ability to act on spermatozoa are still unknown. In order to determine if spermatozoa are targets for estrogens, the presence of estrogen receptors in mature ejaculated spermatozoa has been investigated. The presence of a single isoform of ESR1 (66kDa) and ESR2 (61kDa) was found by Western-blot analysis in samples from seven stallions. Confocal analysi...
Zhang L, Wang X, Zhan Q, Wang Z, Xu M, Zhu D, He F, Liu X, Huang R, Li D, Lei Y, Xie P.Borna disease virus (BDV) is a non-cytolytic, neurotropic RNA virus that can infect many vertebrate species, including humans. To date, BDV infection has been reported in a range of animal species across a broad global geographic distribution. However, a systematic epidemiological survey of BDV infection in domesticated animals in China has yet to be performed. In current study, BDV RNA and antibodies in 2353 blood samples from apparently healthy animals of eight species (horse, donkey, dog, pig, rabbit, cattle, goat, sheep) from three areas in western China (Xinjiang province, Chongqing munic...
Ponthier J, Franck T, Parrilla-Hernandez S, Niesten A, de la Rebiere G, Serteyn D, Deleuze S.Myeloperoxidase (MPO) is a pro-oxidant enzyme associated with decreased motility in thawed equine semen. This study aimed to describe MPO concentration, activity and subunits in raw and thawed semen and to correlate these data with motilities in raw and thawed semen. Semen samples from five stallions were collected four times. Motilities were assessed in raw and thawed semen. MPO assays were performed in raw seminal plasma, raw sperm-rich pellet and thawed semen. Total and active MPO concentrations were, respectively, assayed by enzyme-linked immunosorbent assay and specific immunological extr...
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...
Hdud IM, Mobasheri A, Loughna PT.Chondrocytes are regularly exposed to load-induced stimuli and have the capability to sense and respond to applied mechanical stress. However, the mechanisms involved in chondrocyte mechanotransduction are not clearly understood. The purpose of this study was to explore the effects of cyclic equibiaxial mechanical stretch on the expression of α-BK and TRPV4 channels. Results: Freshly isolated equine articular chondrocytes were subjected to mechanical stress (8% elongation at frequency of 0.5 Hz for 8 h). Western blotting was used to investigate the expression of BKCa and TRPV4 channel prote...
Caselles AB, Miro-Moran A, Morillo Rodriguez A, Gallardo Bolaños JM, Ortega-Ferrusola C, Salido GM, Peña FJ, Tapia JA, Aparicio IM.Apoptosis in the testis is required to ensure an efficient spermatogenesis. However, sometimes, defective germ cells that are marked for elimination during this process escape elimination in the testes, giving rise to ejaculates with increased percentages of abnormal and apoptotic spermatozoa and a high percentage of apoptotic bodies. Apoptosis markers in the ejaculate have been associated with low fertility, either in animals or humans. Therefore, the goal of this study was to investigate whether fresh equine semen contains apoptotic bodies [initially named Merocyanine 540 (M540) bodies] and ...
Williams A, Smith JR, Allaway D, Harris P, Liddell S, Mobasheri A.Arthritic diseases are characterized by the degradation of collagenous and noncollagenous extracellular matrix (ECM) components in articular cartilage. The increased expression and activity of matrix metalloproteinases (MMPs) is partly responsible for cartilage degradation. This study used proteomics to identify inflammatory proteins and catabolic enzymes released in a serum-free explant model of articular cartilage stimulated with the pro-inflammatory cytokine interleukin 1β (IL-1β). Western blotting was used to quantify the release of selected proteins in the presence or absence of the cyc...
Lin YZ, Yang F, Zhang SQ, Sun LK, Wang XF, Du C, Zhou JH.Equine lentivirus receptor 1 (ELR1) has been identified as the sole receptor for equine infectious anemia virus (EIAV) and is a member of the tumor necrosis factor receptor (TNFR) superfamily. In addition to the previously described membrane-associated form of ELR1, two other major alternative splicing variant mRNAs were identified in equine monocyte-derived macrophages (eMDMs). One major spliced species (ELR1-IN) contained an insertion of 153 nt, which resulted in a premature stop codon situated 561 nt upstream of the predicted membrane spanning domain. The other major species (ELR1-DE) has a...
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...
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...
Yeargan MR, Alvarado-Esquivel C, Dubey JP, Howe DK.Equine protozoal myeloencephalitis (EPM) is a debilitating disease of horses caused by Sarcocystis neurona and Neospora hughesi. Sera from 495 horses in Durango State, Mexico were tested for anti-protozoal antibodies using enzyme-linked immunosorbent assays (ELISAs) based on major surface antigens of these two parasites. Antibodies to S. neurona were detected in 240 (48.5%) of the 495 horse sera tested with the rSnSAG2/4/3 trivalent ELISA. Multivariate analysis showed that exposure to S. neurona was associated with age, feeding grains and crops, and small herd size. Antibodies to N. hughesi we...
de Arruda Coutinho LC, de Jesus AL, de Paiva Fontes KF, Coimbra EC, Mariz FC, de Freitas AC, de Cássia Carvalho Maia R, de Castro RS.Equine Infectious Anemia (EIA) is a persistent lentivirus infection of horses which causes a chronic clinical condition with worldwide importance in veterinary medicine. The p26 protein is usually prepared for use as an antigen in serological tests for EIA diagnosis since it is a well-conserved gene sequence and very immunogenic. In view of the ability of yeast to make post-translational modifications of proteins, this study was carried out to allow Pichia pastoris to be used for the expression of a synthetic codon-optimized EIAV p26 gene. The gene was cloned into pPICZαA vector after appropr...
Maischberger E, Cummins CA, Fitzpatrick E, Gallagher ME, Worrall S, Rousseau K, Thornton DJ, Meijer WG, Miranda-Casoluengo R, Duggan VE....In the equine reproductive tract, little is known about mucin gene expression and the role of mucins in barrier function and host-cell interaction. The aims of the study were to identify equine orthologs of mammalian mucin genes using available equine sequence data, to profile expression of equine orthologous mucin genes in the endometrium using reverse transcriptase polymerase chain reaction (RT-PCR), to determine spatial expression patterns of mucin genes using in situ hybridisation, and to confirm the presence of mucin gene products using Western blotting and equine-specific mucin antibodie...
Andersen UV, Howe DK, Dangoudoubiyam S, Toft N, Reinemeyer CR, Lyons ET, Olsen SN, Monrad J, Nejsum P, Nielsen MK.Strongyle parasites are ubiquitous in grazing horses. Strongylus vulgaris, the most pathogenic of the large strongyles, is known for its extensive migration in the mesenteric arterial system. The lifecycle of S. vulgaris is characterised by a long prepatent period where the migrating larvae are virtually undetectable as there currently is no test available for diagnosing prepatent S. vulgaris infection. Presence of S. vulgaris larvae in the arterial system causes endarteritis and thrombosis with a risk of non-strangulating intestinal infarctions. Emergence of anthelmintic resistance among cyat...
Wang L, Luo DK, Pan ZY.The aim of the present study was to investigate the association between glucocorticoid receptors and steroid‑induced avascular necrosis of the femoral head (SANFH). Healthy New Zealand rabbits were randomly divided into 3 groups (n=16/group); the normal (no treatment), control (horse serum injections) and treatment (horse serum and methylprednisolone injections) groups. Methylprednisolone and horse serum injections were used to establish a SANFH model in rabbits; magnetic resonance imaging (MRI) and histopathological analysis were used to evaluate the SANFH rabbit model. Total cholesterol an...
Raabe O, Shell K, Goessl A, Crispens C, Delhasse Y, Eva A, Scheiner-Bobis G, Wenisch S, Arnhold S.Mesenchymal stem cells are regarded as common cellular precursors of the musculoskeletal tissue and are responsible for tissue regeneration in the course of musculoskeletal disorders. In equine veterinary medicine extracorporeal shock wave therapy (ESWT) is used to optimize healing processes of bone, tendon and cartilage. Nevertheless, little is known about the effects of the shock waves on cells and tissues. Thus, the aim of this study was to investigate the influence of focused ESWT on the viability, proliferation, and differentiation capacity of adipose tissue-derived mesenchymal stem cells...
Zamboulis DE, Senior JM, Clegg PD, Gallagher JA, Carter SD, Milner PI.Purinergic pathways are considered important in pain transmission, and P2X receptors are a key part of this system which has received little attention in the horse. The aim of this study was to identify and characterise the distribution of P2X receptor subtypes in the equine digit and associated vasculature and nervous tissue, including peripheral nerves, dorsal root ganglia and cervical spinal cord, using PCR, Western blot analysis and immunohistochemistry. mRNA signal for most of the tested P2X receptor subunits (P2X1-5, 7) was detected in all sampled equine tissues, whereas P2X6 receptor su...
Gutiérrez JM, Tsai WC, Pla D, Solano G, Lomonte B, Sanz L, Angulo Y, Calvete JJ.A polyspecific antivenom is used in Central America for the treatment of envenomings by viperid snakes. This antivenom is generated in horses hyperimmunized with a mixture of venoms from Bothrops asper, Crotalus simus and Lachesis stenophrys. The present study analyzed the ability of this antivenom to neutralize the venoms of three Central American viperid species of the 'Porthidium group', i.e. Porthidium nasutum, Porthidium ophryomegas and Cerrophidion sasai, formerly classified as Cerrophidion godmani. In addition, the immunorecognition of the components of these venoms was assessed by immu...
Swadzba ME, Hauck SM, Naim HY, Amann B, Deeg CA.Complete knowledge of autoantigen spectra is crucial for understanding pathomechanisms of autoimmune diseases like equine recurrent uveitis (ERU), a spontaneous model for human autoimmune uveitis. While several ERU autoantigens were identified previously, no membrane protein was found so far. As there is a great overlap between glycoproteins and membrane proteins, the aim of this study was to test whether pre-enrichment of retinal glycoproteins by ConA affinity is an effective tool to detect autoantigen candidates among membrane proteins. In 1D Western blots, the glycoprotein preparation allow...
Wauters J, Pille F, Martens A, Franck T, Serteyn D, Gasthuys F, Meyer E.Equine joint infection is a life-threatening disorder, and confirmation of the diagnosis can be difficult. Synovial fluid biomarkers may assist the discrimination between infectious and noninfectious joint disease. Objective: This study investigates whether the immunological detection of total and enzymatically active myeloperoxidase (MPO) assists the diagnosis of joint infection in horses. Methods: The following 4 sample groups were included: healthy; osteochondritis dissecans (OCD); traumatic synovitis; and culture-confirmed infected joints. Synovial fluid was analysed for total MPO by a hor...
Altamura G, Strazzullo M, Corteggio A, Francioso R, Roperto F, D'Esposito M, Borzacchiello G.Bovine papillomaviruses (BPVs) types 1 and 2 are the only known papillomaviruses able to jump the species. In fact, BPVs 1/2 induce neoplasia in their natural bovine host but infection is also associated to neoplastic skin lesions in equids termed sarcoids. The equine sarcoid is considered to be the most common equine cutaneous tumour worldwide for which no effective therapy is available. Very little is known about the molecular mechanisms underlying tumourigenesis, although genes contributing to sarcoid development have been identified. Several studies associate the development of cancer to t...
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 ...
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...
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...
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...
Burk SV, Dangoudoubiyam S, Brewster-Barnes T, Bryant UK, Howe DK, Carter CN, Vanzant ES, Harmon RJ, Kazacos KR, Rossano MG.Currently, diagnosis of Parascaris equorum infection in equids is limited to patent infections. The goals of this study were to culture P. equorum larvae in vitro and identify excretory-secretory (ES) products for prepatent diagnostic testing. Parascaris equorum L2/L3 larvae were hatched and cultured for up to 3 weeks for ES product collection. Fifth stage (L5) P. equorum were also cultured for ES product collection. Examination of ES fractions by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver stain revealed L2/L3 products ranging from 12-94 kDa and L5 products...
Arkoun B, Gautier C, Delalande C, Barrier-Battut I, Guénon I, Goux D, Bouraïma-Lelong H.Among mammals, the stallion produces the largest amount of testicular estrogens. These steroid hormones are produced mainly by Leydig and Sertoli cells in the testis and also in the epididymis. Their role in horse testicular physiology and their ability to act on spermatozoa are still unknown. In order to determine if spermatozoa are targets for estrogens, the presence of estrogen receptors in mature ejaculated spermatozoa has been investigated. The presence of a single isoform of ESR1 (66kDa) and ESR2 (61kDa) was found by Western-blot analysis in samples from seven stallions. Confocal analysi...
Brandon CI, Heusner GL, Caudle AB, Fayrer-Hosken RA.The objectives of this study were to 1) identify proteins found in stallion seminal plasma utilizing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in conjunction with Western blot analysis; and 2) to determine if any of these individual proteins were correlated with stallion fertility utilizing regression analysis. Fertility was quantified by assigning a breeding score for each stallion. Each score was calculated by dividing the number of conceptions by the number of breedings for each stallion for four successive breeding seasons (1992-1995). Ejaculates from stallions of known ...
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...
Huang X, Xuan X, Yokoyama N, Xu L, Suzuki H, Sugimoto C, Nagasawa H, Fujisaki K, Igarashi I.The gene encoding a truncated merozoite antigen-2 (EMA-2t) of Babesia equi was cloned and highly expressed in Escherichia coli as a glutathione S-transferase fusion protein (G-rEMA-2t). Both G-rEMA-2t and rEMA-2t (after the removal of glutathione S-transferase) had good antigenicity. Either Western blot analysis with rEMA-2t or enzyme-linked immunosorbent assay (ELISA) with G-rEMA-2t clearly discriminated the sera of horses experimentally infected with B. equi from sera of horses infected with Babesia caballi and healthy horses, although rEMA-2t was not suitable for ELISA, probably owing to it...
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...
Sato F, Kanno T, Nagasawa S, Yanaihara N, Ishida N, Hasegawa T, Iwanaga T.We investigated the existence of chromogranin A (CgA) in salivary glands of the horse by Western blotting and enzyme immunoassay (EIA) using an antiserum against a peptide sequence of equine CgA. We also compared its cellular distribution between the horse and rat salivary glands with a tyramide signal amplification immunofluorescence technique. Western blotting gave three significant immunoreactive bands (74, 56 and 48 kDa) in adrenal medulla and three major salivary glands of horses. Immunoreactivities for CgA measured by EIA in horses were 154.05 +/- 41.46, 20.32 +/- 5.59 and 4.43 +/- 2.23 ...
Rivero JL, Talmadge RJ, Edgerton VR.In adult horses, three myosin heavy chain (MyHC) isoforms can be identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunohistochemistry using specific anti-MyHC monoclonal antibodies. This report studies the suitability of a consistent SDS-PAGE technique for quantifying MyHC profiles in homogenized cryostate sections of equine gluteus medius muscle biopsies (n = 18). The method used (previously described by R. J. Talmadge and R. R. Roy; J. Appl. Physiol. 1993, 75, 2337-2340) resolved MyHCs in three bands: I, IIB or IIX, and IIA from the fastest to the slowe...
Bullido R, Doménech N, Alvarez B, Alonso F, Babín M, Ezquerra A, Ortuño E, Domínguez J.A set of five monoclonal antibodies (mAb) against porcine major histocompatibility complex (MHC), or swine leukocyte antigens (SLA), class II molecules has been characterized. These mAbs appear to recognize monomorphic determinants on SLA-DR (2F4, 1F12 and 2E9/13) and SLA-DQ (BL2H5 and BL4H2) molecules, as assessed by flow cytometry and immunoprecipitation. By Western blot, the 2F4, 1F12, BL2H5 and BL4H2 epitopes were located on the beta-chains of these molecules. mAbs 2F4 and 1F12 crossreact with leucocytes of dog, cattle, horse and human; mAbs 2E9/13, BL2H5 and BL4H2 bind leucocytes of cattl...
Ibrahim S, Steinbach F.Earlier studies investigating the cross-reactivity of antibodies submitted to the HLDA8 had used flow cytometry as a method of choice to screen mAbs for reactivity with equine leukocytes, including two-color flow-cytometry to characterize the lymphocyte population they detect. In addition, immuno-histochemistry (IHC) was used to detect distribution of positive cells in lymphoid tissue sections. In this study we performed immunoprecipitation (IP) to complement the previous results and add valuable information regarding the molecules detected by the cross-reacting antibodies. Surface molecules f...
Wang L, Luo DK, Pan ZY.The aim of the present study was to investigate the association between glucocorticoid receptors and steroid‑induced avascular necrosis of the femoral head (SANFH). Healthy New Zealand rabbits were randomly divided into 3 groups (n=16/group); the normal (no treatment), control (horse serum injections) and treatment (horse serum and methylprednisolone injections) groups. Methylprednisolone and horse serum injections were used to establish a SANFH model in rabbits; magnetic resonance imaging (MRI) and histopathological analysis were used to evaluate the SANFH rabbit model. Total cholesterol an...
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...
Blythe LL, Granstrom DE, Hansen DE, Walker LL, Bartlett J, Stamper S.To determine seroprevalence of antibodies to Sarcocystis neurona in neurologically normal horses residing in 4 regions of Oregon and to describe the effects of age, gender, breed, and housing on seroprevalence within each region. Methods: Prevalence survey. Methods: Serum samples from 334 horses systematically selected by practicing veterinarians. Methods: Antibodies to S neurona were measured in sera, using a western blot. Information including age, gender, breed, housing, geographic location, and duration of residence was obtained for each horse. Data were analyzed, using descriptive statist...
Sosnicki AA, Lutz GJ, Rome LC, Goble DO.Until now, there has been no reliable method for histochemical determination of fiber types of single skinned muscle fibers. The major problem arises from the fact that most histochemical techniques use cross-sections of a large group of fibers and compare a given fiber with those surrounding it. This is not possible with a single skinned fiber which has been separated from a bundle to be used for mechanical analysis. A further problem is that the skinning procedure itself may alter the staining pattern. We have developed a procedure by which multiple cross-sections of single skinned fibers ca...
Grignard E, Morin J, Vernet P, Drevet JR.We report here on the cloning of cDNAs coding bovine and equine orthologs of mouse epididymis-restricted and sperm-bound glutathione peroxidase 5 (GPX5), a selenium-independent member of the multigenic GPX family in mammals. The complete sequence of bovine GPX5 as well as a partial sequence of the equine GPX5 were characterized, conceptually translated and aligned with other known mammalian GPX5 proteins. Using Northern blotting assays, we show that the level of expression of GPX5 is high in bovine but low in equine and that in both species the regionalization of GPX5 expression in epididymis ...
Aquino LP, Machado RZ, Lemos KR, Marques LC, Garcia MV, Borges GP.The present research investigated the presence of T. evansi antibodies in animals from the subregion of Nhecolandia, in the Pantanal Sul-mato-grossense, by means of an enzyme linked immunosorbent assay (ELISA) and indirect immunofluorescence antibody test (IFAT), and the pattern of polypeptide recognition by sera from experimentally and naturally infected hosts using Western blotting. Serum samples were obtained from bovines (n = 102), horses (n = 98), and dogs (n = 55), and from 32 free-ranging coatis (Nasua nasua). None of the bovines were found positive, while sera from 16 dogs (29%) and 23...
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...
Cabre O, Durand JP, Prangé A, Gomez J, Maurizi L, Tolou H, Davoust B.This study was carried out in 2003 to detected serological evidence of West Nile virus infection in 190 Army horses kept nearby French troops stationed in Southeast France and in Africa (Chad, Côte d'Ivoire and Senegal). Both IgG and IgM antibodies were searched for using an ELISA assay. Specifiity of IgG antibodies was determined by western blot and plaque reduction seroneutraization. Finding showed that 79% of the Army horses (n=96) tested in Africa presented specific IgG antibodies. All horses that were seropositive for IgG were seronegative for IgM. None of the Army horses (n=94) tested i...
Annandale EJ, Valberg SJ, Mickelson JR, Seaquist ER.Equine polysaccharide storage myopathy (PSSM) is an inherited disorder characterized by the accumulation of glycogen and abnormal polysaccharide in muscle with normal glyco(geno)lytic enzyme activities. The purpose of this study was to evaluate in vivo insulin sensitivity and glucose excursion in PSSM using a euglycemic hyperinsulinemic clamp. In addition, the content of muscle glucose transporters (GLUT1 and GLUT4) and the insulin receptor was determined in muscle biopsies using Western blot analysis. The glycogen content was 1.8-fold higher, and isolated polysaccharide analyzed by iodine abs...
Zhang L, Wang X, Zhan Q, Wang Z, Xu M, Zhu D, He F, Liu X, Huang R, Li D, Lei Y, Xie P.Borna disease virus (BDV) is a non-cytolytic, neurotropic RNA virus that can infect many vertebrate species, including humans. To date, BDV infection has been reported in a range of animal species across a broad global geographic distribution. However, a systematic epidemiological survey of BDV infection in domesticated animals in China has yet to be performed. In current study, BDV RNA and antibodies in 2353 blood samples from apparently healthy animals of eight species (horse, donkey, dog, pig, rabbit, cattle, goat, sheep) from three areas in western China (Xinjiang province, Chongqing munic...
Nevalainen M, Raulo SM, Brazil TJ, Pirie RS, Sorsa T, McGorum BC, Maisi P.We report the effects of mouldy hay/straw exposure, inhaled hay dust suspension (HDS) and inhaled lipopolysaccharide (LPS) on bronchoalveolar lavage fluid (BALF) gelatinolytic matrix metalloproteinase (MMP) levels and degree of activation in healthy (n = 6) and heaves- (previously termed chronic obstructive pulmonary disease) affected (n = 6 or 7) horses. Gelatinolytic MMPs in BALF were quantified by zymography, and gelatinases were shown by Western immunoblotting to be MMP-2 and MMP-9. Hay/straw and HDS challenges increased BALF total gelatinolytic activity only in heaves horses, with the maj...
Chang Y, Novosol V, McDonough SP, Chang CF, Jacobson RH, Divers T, Quimby FW, Shin S, Lein DH.Eight 1-year-old ponies were vaccinated with recombinant OspA (ospA gene derived from B. burgdorferi B31) with adjuvant (aluminium hydroxide). Four ponies were used as non-vaccinated controls with adjuvant. One hundred and twelve days after the first vaccination, the vaccinated and non-vaccinated ponies were challenged by exposure to B. burgdorferi-infected adults tick (Ixodes scapularis) collected from Westchester County, New York (tick infection rate >/=60%). Protection from infection was evaluated by culture for B. burgdorferi from three monthly skin biopsies taken near the site of tick bit...
Go YY, Snijder EJ, Timoney PJ, Balasuriya UB.Equine arteritis virus (EAV) replicase consists of two polyproteins (pp1a and pp1ab) that are encoded by open reading frames (ORFs) 1a and 1b of the viral genome. These two replicase polyproteins are posttranslationally processed by three ORF 1a-encoded proteinases to yield at least 13 nonstructural proteins (nsp1 to nsp12, including nsp7α and 7β). These nsps are expressed in EAV-infected cells, but the equine immune response they induce has not been studied. Therefore, the primary purpose of this study was to evaluate the humoral immune response of horses to each of the nsps following EAV i...
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...
Crossett B, Suire S, Herrler A, Allen WR, Stewart F.One of the major, progesterone-dependent proteins secreted into the uterine lumen of the mare is a 19-kDa lipocalin (P19). It associates strongly with the embryonic capsule that envelops the young horse conceptus in early gestation, suggesting that it may be involved in sustaining early development. However, it was not known whether the protein was transported through the capsule and/or trophoblast layer and into the yolk sac cavity. To address this question, polyclonal antisera were raised against a C-terminal peptide (based on the deduced amino acid sequence of P19) and a recombinant-derived...
Bell CW, Boyle DB, Whalley JM.Transcript mapping of the equine herpesvirus 1 (EHV-1) glycoprotein B (gB) gene homologue by Northern blot, S1 nuclease and primer extension analyses indicated that two overlapping transcripts of 3.4 and 4.6 kb originated from the same strand and were transcribed from left to right between coordinates 0.40 and 0.43 of the EHV-1 genome. The 3.4 kb transcript encoded EHV-1 gB and the 5' RNA terminus was located approximately 30 bases downstream from a probable TATA element. The coding region of the gB gene homologue was reconstructed from two subclones using oligonucleotide mutagenesis and inser...
Pfeifer Barbosa I, Böse R, Peymann B, Friedhoff KT.Epidemiological studies of Babesia equi and B. caballi were undertaken in a herd of 120 pastured horses in Rio de Janeiro, Brazil. The area where the horses were held was shown to be highly endemic for both Babesia spp., i.e. the prevalence of B. equi antibodies in horses aged 6 months or older ranged from 90.6% to 100% as determined by the immunofluorescence antibody (IFA) test, and the prevalence of B. caballi antibodies as determined by Western blot ranged from 59.4% to 65.5%. From the herd, 20 foals and their dams were selected to estimate the degree of tick infestation and the foals were ...
