Immunoblotting, also known as Western blotting, is a laboratory technique used to detect specific proteins in a sample. In equine research, this method is applied to analyze protein expression and modifications in horses, contributing to the understanding of various biological processes and disease states. Immunoblotting involves the separation of proteins by gel electrophoresis, followed by their transfer to a membrane and subsequent detection using specific antibodies. This technique is utilized in studies investigating equine physiology, pathology, and immunology, providing insights into protein-related functions and interactions. This page compiles peer-reviewed research studies and scholarly articles that explore the application, methodology, and findings of immunoblotting in equine science.
Dyrda K, Orzołek A, Ner-Kluza J, Wysocki P.Phosphorylation and dephosphorylation of proteins are considered to be the most important processes in sperm maturation during epididymal transit. The main aim of this study was to isolate and identify phosphoproteins from the epididymal milieu obtained from reproductively mature stallions during and out of the breeding season. With the use of 1D-PAGE and nanoLC-MS/MS, we identified phosphoproteins that fulfil various functions: regulatory, transport, motility, ubiquitination, chaperone, antioxidant, apoptotic and enzymatic. Moreover, we characterized tyrosine, serine and threonine phosphoryla...
Autry JM, Karim CB, Cocco M, Carlson SF, Thomas DD, Valberg SJ.We have analyzed protein expression and enzyme activity of the sarcoplasmic reticulum Ca2+-transporting ATPase (SERCA) in horse gluteal muscle. Horses exhibit a high incidence of recurrent exertional rhabdomyolysis, with myosolic Ca2+ proposed, but yet to be established, as the underlying cause. To better assess Ca2+ regulatory mechanisms, we developed an improved protocol for isolating sarcoplasmic reticulum (SR) vesicles from horse skeletal muscle, based on mechanical homogenization and optimized parameters for differential centrifugation. Immunoblotting identified the peak subcellular fract...
Stokes SM, Burns TA, Watts MR, Bertin FR, Stefanovski D, Medina-Torres CE, Belknap JK, van Eps AW.Continuous digital hypothermia (CDH) prevents lamellar failure in the euglycemic hyperinsulinemic clamp (EHC) model of laminitis, but the protective mechanisms are unclear. Objective: To determine if CDH inhibits lamellar inflammatory signaling in the EHC model of laminitis. Methods: Eight Standardbred horses. Methods: Prospective experimental study. Horses underwent an EHC, with 1 forelimb treated with CDH and the other kept at ambient temperature (AMB). Horses were euthanized 48 hours after initiation of the EHC and lamellar tissue was analyzed via polymerase chain reaction (pro-inflammato...
Jin C, Cherian RM, Liu J, Playà-Albinyana H, Galli C, Karlsson NG, Breimer ME, Holgersson J.Animal bioprosthetic heart valves (BHV) are used to replace defective valves in patients with valvular heart disease. Especially young BHV recipients may experience a structural valve deterioration caused by an immune reaction in which α-Gal and Neu5Gc are potential target antigens. The expression of these and other carbohydrate antigens in animal tissues used for production of BHV was explored. Protein lysates of porcine aortic and pulmonary valves, and porcine, bovine and equine pericardia were analyzed by Western blotting using anti-carbohydrate antibodies and lectins. N-glycans were relea...
Equine infectious anaemia (EIA) is a blood borne disease that is listed among the notifiable diseases of the World Organisation for Animal Health (OIE). EIA is also regulated by the OIE for the international trading provisions and is generally subject to control programmes. Since 2011, Italy has been conducting a surveillance plan based on a three-tier diagnostic system, using a serological ELISA as screening test, an agar gel immunodiffusion test (AGIDT) as a confirmatory method, and an immunoblot (IB) as an alternative confirmatory assay for discordant results between the first two tests. As...
Wise LN, Kappmeyer LS, Silva MG, White SN, Grause JF, Knowles DP.Certain countries including the United States remain non-endemic for particular infectious diseases such as equine piroplasmosis through import restrictions and surveillance. Endemic regions often employ premunition as the primary method to control disease, however in non-endemic countries, chemosterilization combined with methods to confirm parasite elimination are required to maintain disease-free status. The ability of imidocarb diproprionate (ID) to clear persistent Theileria equi infection from infected horses has been shown through the inability of treated horses to transmit via blood tr...
Dohre SK, Kamthan A, Singh S, Alam SI, Kumar S.Glanders is a disease of horses, donkeys and mules. The causative agent Burkholderia mallei, is a biorisk group 3 pathogen and is also a biothreat agent. Simple and rapid diagnostic tool is essential for control of glanders. Using a proteomic approach and immunoblotting with equine sera, we identified 12 protein antigens that may have diagnostic potential. Various immunoreactive proteins e.g. GroEL, translation elongation factor Tu, elongation factor Ts, arginine deiminase, malate dehydrogenase, DNA directed RNA polymerase subunit alpha were identified on 2-dimentional immunoblots. One of thes...
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...
Campolo A, de Laat MA, Keith L, Gruntmeir KJ, Lacombe VA.Insulin dysregulation is common in horses although the mechanisms of metabolic dysfunction are poorly understood. We hypothesized that insulin signaling in striated (cardiac and skeletal) muscle and lamellae may be mediated through different receptors as a result of receptor content, and that transcriptional regulation of downstream signal transduction and glucose transport may also differ between tissues sites during hyperinsulinemia. Archived samples from horses treated with a prolonged insulin infusion or a balanced electrolyte solution were used. All treated horses developed marked hyperin...
Galvão CM, Rezende-Gondim MM, Chaves AC, Schares G, Ribas JR, Gondim LF.Donkeys (Equus asinus) are closely related to horses and are known to be infected by several equine pathogens. Neospora caninum and Neospora hughesi are protozoan parasites that infect horses, but they were not confirmed in donkeys up to this date. The aim of this study was to evaluate the exposure of donkeys (Equus asinus) to Neospora spp. using tachyzoites of N. caninum as antigen and employing two common serologic methods, IFAT and immunoblot. Sera from 500 donkeys were obtained from 30 municipalities in Bahia state and tested by IFAT. Two of 500 sera were positive for Neospora spp. by IFAT...
Kullmann A, Weber PS, Bishop JB, Roux TM, Norby B, Burns TA, McCutcheon LJ, Belknap JK, Geor RJ.Hyperinsulinaemia is implicated in the pathogenesis of endocrinopathic laminitis. Insulin can bind to different receptors: two insulin receptor isoforms (InsR-A and InsR-B), insulin-like growth factor-1 receptor (IGF-1R) and InsR/IGF-1R hybrid receptor (Hybrid). Currently, mRNA expression of these receptors in equine tissues and the influence of body type and dietary carbohydrate intake on expression of these receptors is not known. Objective: The study objectives were to characterise InsR-A, InsR-B, IGF-1R and Hybrid expression in lamellar tissue (LT) and insulin responsive tissues from horse...
Douglas HF, Southwood LL, Meyer-Ficca ML, Hart SK, Meyer RG.To evaluate the poly (ADP-ribose) polymerase-1 (PARP1) enzyme and its inhibition in horses and explore its potential as a novel therapeutic target for equine intestinal ischemia-reperfusion injury by (1) identifying poly (ADP-ribose) (PAR) as an indication of PARP1 activation in equine cells using available immunoblot analytical techniques, (2) inducing PARP1 activation in an in vitro oxidative DNA damage model, (3) and demonstrating the inhibition of PARP1 in equine cells using commercially available PARP1 inhibitors. Methods: Experimental study. Methods: Blood samples were collected from sys...
Dryburgh EL, Marsh AE, Dubey JP, Howe DK, Reed SM, Bolten KE, Pei W, Saville WJ.Sarcocystis neurona is considered the major etiologic agent of equine protozoal myeloencephalitis (EPM), a neurological disease in horses. Raccoon ( Procyon lotor ) is considered the most important intermediate host in the life cycle of S. neurona in the United States; S. neurona sarcocysts do mature in raccoon muscles, and raccoons also develop clinical signs simulating EPM. The focus of this study was to determine if sarcocysts would develop in raccoons experimentally inoculated with different host-derived strains of in vitro-cultivated S. neurona merozoites. Four raccoons were inoculated wi...
Canisso IF, Ball BA, Scoggin KE, Squires EL, Williams NM, Troedsson MH.The objectives of this study were to: (i) determine alpha-fetoprotein (AFP) concentrations in fetal fluids (FF), and (ii) compare plasma concentrations of AFP in mares with placentitis (n=17) and gestationally age-matched control mares (n=17). Fetal fluid sampling (FFS, n=7/group) was performed at 0, 5 and 12 days post inoculation (DPI) or until abortion. Plasma was harvested daily for 12 days or until abortion. Placentitis was induced via intracervical inoculation of Streptococcus equi ssp. zooepidemicus. Proteins present in the FF were resolved by 1D-SDS-PAGE, and immunoblotting was used to ...
de Laat MA, Clement CK, Sillence MN, McGowan CM, Pollitt CC, Lacombe VA.An increased incidence of metabolic disease in horses has led to heightened recognition of the pathological consequences of insulin resistance. Laminitis, failure of the weightbearing digital lamellae, is an important consequence. Altered trafficking of specialised glucose transporters (GLUTs), responsible for glucose uptake, is central to the dysregulation of glucose metabolism and may play a role in the pathophysiology of laminitis. Objective: We hypothesised that prolonged hyperinsulinaemia alters the regulation of glucose transport in insulin-sensitive tissue and digital lamellae. Our obje...
Sguazza GH, Fuentealba NA, Tizzano MA, Galosi CM, Pecoraro MR.Equine influenza virus is a leading cause of respiratory disease in horses worldwide. Disease prevention is by vaccination with inactivated whole virus vaccines. Most current influenza vaccines are generated in embryonated hens' eggs. Virions are harvested from allantoic fluid and chemically inactivated. Although this system has served well over the years, the use of eggs as the substrate for vaccine production has several well-recognized disadvantages (cost, egg supply, waste disposal and yield in eggs). The aim of this study was to evaluate a baculovirus system as a potential method for prod...
Mahmoud HY, Andoh K, Hattori S, Terada Y, Noguchi K, Shimoda H, Maeda K.In this study, we attempted to express twelve glycoproteins of equine herpesvirus-1 (EHV-1) in 293T cells and to characterize these using monoclonal antibodies (MAbs) and horse sera against EHV-1. Expression of glycoprotein B (gB), gC, gD, gG, gI and gp2 was recognized by immunoblot analysis using horse sera, but that of gE, gH, gK, gL, gM and gN was not. Four MAbs recognized gB, four recognized gC and one recognized gp2. Two MAbs against gB cross-reacted with EHV-4. Interestingly, coexpression of gE and gI and gM and gN enhanced their antigenicity. Furthermore, immunoblot analysis of gp2 show...
Donnelly KS, Giuliano EA, Sharma A, Tandon A, Rodier JT, Mohan RR.To explore (i) the potential of polyethylenimine (PEI) nanoparticles as a vector for delivering genes into equine corneal fibroblasts (ECFs) using green fluorescent protein (GFP) marker gene, (ii) whether PEI nanoparticle-mediated decorin (DCN) gene therapy could be used to inhibit fibrosis in the equine cornea using an in vitro model. Methods: Polyethylenimine-DNA nanoparticles were prepared at nitrogen-to-phosphate (N-P) ratio of 15 by mixing 22 kDa linear PEI and a plasmid encoding either GFP or DCN. ECFs were generated from donor corneas as previously described. Initially, GFP was introduc...
Szober CM, Hauck SM, Euler KN, Fröhlich KJ, Alge-Priglinger C, Ueffing M, Deeg CA.The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses' vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, an...
Lasko J, Schlingmann K, Klocke A, Mengel GA, Turner R.In spite of the importance of sperm motility to fertility in the stallion, little is known about the signaling pathways that regulate motility in this species. In other mammals, calcium/calmodulin signaling and the cyclic AMP/protein kinase-A pathway are involved in sperm motility regulation. We hypothesized that these pathways also were involved in the regulation of sperm motility in the stallion. Using immunoblotting, calmodulin and the calmodulin-dependent protein kinase II β were shown to be present in stallion sperm and with indirect immunofluorescence calmodulin was localized to the acr...
Khan I, Wieler LH, Melzer F, Gwida M, Santana VL, de Souza MM, Saqib M, Elschner MC, Neubauer H.The sensitivity and specificity of three commercially available complement fixation test (CFT) antigens from c.c.pro (c.c.pro), Central Veterinary Institute of Wageningen UR (CIDC) and the United States Department of Agriculture (USDA) were comparatively evaluated by testing 410 sera collected from glanders-endemic and non-endemic areas (200 true-negative randomly collected sera and 210 sera collected from experimentally immunised animals (12 rabbits, 19 horses), clinically positive (135) and culture-positive (44) horses, donkeys and mules). Immunoblotting (IB) was used as the gold standard te...
Almeida J, Ball BA, Conley AJ, Place NJ, Liu IK, Scholtz EL, Mathewson L, Stanley SD, Moeller BC.Anti-Müllerian hormone (AMH), a member of the transforming growth factor β superfamily of growth and differentiation factors, is expressed in granulosa cells of preantral and small antral ovarian follicles. In humans, AMH appeared to regulate recruitment and growth of small ovarian follicles. Furthermore, circulating AMH concentrations were elevated in women with granulosa-cell tumors (GCT). In the horse, GCTs are the most common tumor of the ovary, and a variety of endocrine assays have been used to diagnose presumptive GCTs. The objectives of the present study were to validate a heterologo...
Liccardi G, Dente B, Restani P, Senna G, Falagiani P, Ballabio C, D'Amato G.In this report we describe un unusual case of exclusive allergic sensitization to furry animals, as a possible study model to speculate about different modalities ofsensitization to allergens of common and less common mammalian species. A 27-year-old woman referred in our Allergological Centre for the occurrence of conjunctival and severe respiratory symptoms after contact with several animals such as cats, dogs, rabbits, horses, cows etc. Patient underwent clinical and anamnestic evaluation including a detailed information on the modality of exposure to different furry animals. Skin-prick-tes...
Rodrigues NL, Doré M, Doucet MY.To characterize the expression of the cyclooxygenase (COX)-1 and COX-2 isoforms in naturally occurring ulcers of the nonglandular portion of the stomach in horses. SPECIMEN POPULATION: 38 specimens from ulcerated stomachs and 10 specimens from healthy stomachs. Methods: Specimens were collected at an abbatoir; for each specimen of squamous gastric mucosa, 1 portion was fixed in neutral-buffered 10% formalin for immunohistochemical analysis and another was frozen at -70 degrees C for immunoblotting analysis. Immunoreactivity to 2 antibodies, MF241 (selective for COX-1) and MF243 (selective for ...
Desantis S, Ventriglia G, Zizza S, Guaricci AC, Losurdo M, Zarrilli A, Albrizio M.The presence of the mu-opioid receptor (MOR) was investigated in the mare oviduct during oestrus and anoestrus, by means of immunoblotting and immunohistochemistry. Immunoblotting analysis showed that the MOR protein is expressed as 65, 50 and 30 kDa forms in the infundibulum and ampulla both in oestrus and anoestrus, while the 30 kDa form is absent in the isthmus. Moreover, different levels of expression were observed along the ampulla in the two periods examined. Immunohistochemistry revealed MOR in the mucosal epithelium, stromal cells, myocytes and blood vessels. Ciliated cells expressed M...
Barbey C, Budin-Verneuil A, Cauchard S, Hartke A, Laugier C, Pichereau V, Petry S.Rhodococcus equi is one of the most important causes of mortality in foals between 1 and 6 months of age. Although rare, infection also occurs in a variety of other mammals including humans, often following immunosuppression of various causes. Secreted proteins are known to mediate important pathogen-host interactions and consequently are favored candidates for vaccine development as they are the most easily accessible microbial antigens to the immune system. Here, we describe the results of a proteomic analysis based on SDS-PAGE, immunoblot and mass spectrometry, which was carried out aiming ...
Desantis S, Albrizio M, Ventriglia G, Deflorio M, Guaricci AC, Minoia R, De Metrio G.The presence of the mu-opioid receptor and the type of glycosylation in the third extra-cellular loop of this receptor was investigated in the isthmus of mare oviduct during oestrus by means of immunoblotting and immunohistochemistry combined with enzymatic (N-glycosidase F and O-glycosidase) and chemical (beta-elimination) treatments. Immunoblotting analysis showed that the mu-opioid receptor consists of two peptides with molecular weights of around 65 and 50 kDa. After N-deglycosylation with N-glycosidase F an additional immunoreactive peptide was observed at around 30 KDa. The cleavage of O...
Dell'Aquila ME, Albrizio M, Guaricci AC, De Santis T, Maritato F, Tremoleda JL, Colenbrander B, Guerra L, Casavola V, Minoia P.The micro-opioid receptor (MOR) was identified in equine oocytes, cumulus and granulosa cells. By RT-PCR, a 441bp fragment was observed. By immunoblotting, a 65 kDa band was detected in samples of winter anestrous whereas in cells recovered in breeding season, two bands, 65 and 50 kDa, were found. The 65 kDa band was significantly more intense in winter anestrous specimens. In samples recovered in the breeding season, this band significantly decreased with the raise of follicle size and was heavier in compact oocytes and cumulus cells. The protein was localized on the oolemma and within the cy...
Ortega-Ferrusola C, Sotillo-Galán Y, Varela-Fernández E, Gallardo-Bolaños JM, Muriel A, González-Fernández L, Tapia JA, Peña FJ.The kinematics of the appearance of apoptotic markers was studied by flow cytometry and immunoblot assays in equine spermatozoa subjected to freezing and thawing. Caspase activity, low mitochondrial membrane potential, and increases in sperm membrane permeability were observed in all of the phases of the cryopreservation procedure. Freezing and thawing caused an increase in membrane permeability and changes in the pattern of caspase activity; decreases in mitochondrial membrane potential were observed after centrifugation and cooling to 4 degrees C and after freezing and thawing. It is propose...
Nic An Ultaigh S, Ryan MF.Proteinases released during the in vitro maintenance of asynchronous cultures of the free-living nematode Caenorhabditis elegans were characterized on the basis of subunit composition, fluorogenic substrate specificity, inhibitor sensitivity and pH optima. Cysteine proteinases are present in the excretory-secretory products (ESP) as indicated by the hydrolysis of cathepsin fluorogenic substrates and confirmed by immunoblotting. Serine proteinases were predominant as indicated by substrate gel analysis and inhibitor studies. The presence of metallo-proteinases was also indicated by inhibitor st...
Takai S, Koike K, Ohbushi S, Izumi C, Tsubaki S.Antigens of Rhodococcus equi were analyzed by immunoblotting with naturally infected foal sera. Immunoblots of whole-cell antigen preparations of clinical isolates of R. equi revealed that major protein bands with molecular masses of 15 to 17 kDa were present in all clinical isolates tested and all isolates virulent for mice. In contrast, the 15- to 17-kDa antigens were not identified by immunoblotting in ATCC 6939, a type strain of R. equi that was avirulent for mice. Whole-cell antigens of 102 environmental isolates were investigated by immunoblotting and the mouse pathogenicity test. Twenty...
Ott DE, Coren LV, Sowder RC, Adams J, Nagashima K, Schubert U.Some retroviruses contain monoubiquitinated Gag and do not bud efficiently from cells treated with proteasome inhibitors, suggesting an interaction between the ubiquitin-proteasome system and retrovirus assembly. We examined equine infectious anemia virus (EIAV) particles and found that approximately 2% of the p9(Gag) proteins are monoubiquitinated, demonstrating that this Gag protein interacts with an ubiquitinating activity. Different types of proteasome inhibitors were used to determine if proteasome inactivation affects EIAV release from chronically infected cells. Pulse-chase immunoprecip...
Ortega-Ferrusola C, Sotillo-Galán Y, Varela-Fernández E, Gallardo-Bolaños JM, Muriel A, González-Fernández L, Tapia JA, Peña FJ.The kinematics of the appearance of apoptotic markers was studied by flow cytometry and immunoblot assays in equine spermatozoa subjected to freezing and thawing. Caspase activity, low mitochondrial membrane potential, and increases in sperm membrane permeability were observed in all of the phases of the cryopreservation procedure. Freezing and thawing caused an increase in membrane permeability and changes in the pattern of caspase activity; decreases in mitochondrial membrane potential were observed after centrifugation and cooling to 4 degrees C and after freezing and thawing. It is propose...
McDonald TL, Larson MA, Mack DR, Weber A.Mammary-associated serum amyloid A 3 (M-SAA3) was secreted at highly elevated levels in bovine, equine and ovine colostrum and found at lower levels in milk 4 days postparturition. N-terminal sequencing of the mature M-SAA3 protein from all the three species revealed a conserved four amino acid motif (TFLK) within the first eight residues. This motif has not been reported to be present in any of the hepatically-produced acute phase SAA (A-SAA) isoforms. Cloning of the bovine M-Saa3 cDNA from mammary gland epithelial cells revealed an open reading frame that encoded a precursor protein of 131 a...
Södersten F, Ekman S, Schmitz M, Paulsson M, Zaucke F.Injuries of the equine superficial digital flexor tendon are common in racing horses. Knowledge of the tendon matrix composition is crucial to understand physiological and pathological processes in the tendon. The aim of this study was to analyze TSP-4 expressed in equine tendon. Equine tendons were extracted with 10 mM EDTA-containing buffer and TSP-4 purified with ion-exchange chromatography followed by heparin affinity chromatography. The purified TSP-4 was analyzed by one- and two-dimensional SDS-PAGE, immunoblotting, and MALDI-TOF mass spectrometry. Purified TSP-4 gave bands reacting with...
Rwambo PM, Issel CJ, Adams WV, Hussain KA, Miller M, Montelaro RC.Three ponies were inoculated with plasma containing 10(4.8) TCID50 of equine infectious anemia virus (EIAV) and observed for 165 to 440 days. Each pony developed a febrile response within 3 weeks of infection during which a plasma viremia greater than or equal to 10(3.5) TCID50/ml was observed. Analyses of four isolates from sequential febrile episodes in a single pony were conducted by two-dimensional tryptic peptide maps and with monoclonal antibodies in immunoblots. Structural and antigenic alterations were observed in the envelope glycoproteins gp90 and gp45, with greatest variation in gp9...
MacLachlan NJ, Balasuriya UB, Hedges JF, Schweidler TM, McCollum WH, Timoney PJ, Hullinger PJ, Patton JF.Equine arteritis virus (EAV) is the causative agent of equine viral arteritis, an apparently emerging disease of equids. In this study, the antibody response of horses to the structural proteins of EAV was evaluated using gradient-purified EAV virions and baculovirus-expressed recombinant EAV structural proteins (G(L), G(S), M, N) as antigens in a Western immunoblotting assay. Thirty-three sera from horses that previously had been naturally or experimentally infected with EAV were evaluated, including samples from mares, geldings, and both persistently and nonpersistently infected stallions. S...
Chaichanasiriwithaya W, Rikihisa Y, Yamamoto S, Reed S, Crawford TB, Perryman LE, Palmer GH.Ehrlichia risticii causes an acute infectious disease in horses called Potomac horse fever. To investigate the biological diversity of E. risticii organisms, nine E. risticii isolates derived from the peripheral blood monocytes of clinically sick horses in Ohio and Kentucky during the summers of 1991 and 1993 were compared with Illinois and Virginia isolates originally obtained from horses in Maryland in 1984. Seven of the nine isolates (081, 606, 380, 679, As, Co, and Ov) formed large morulae (tightly packed inclusions of ehrlichial organisms). The remaining isolates, including 1984 isolates,...
Donnelly KS, Giuliano EA, Sharma A, Tandon A, Rodier JT, Mohan RR.To explore (i) the potential of polyethylenimine (PEI) nanoparticles as a vector for delivering genes into equine corneal fibroblasts (ECFs) using green fluorescent protein (GFP) marker gene, (ii) whether PEI nanoparticle-mediated decorin (DCN) gene therapy could be used to inhibit fibrosis in the equine cornea using an in vitro model. Methods: Polyethylenimine-DNA nanoparticles were prepared at nitrogen-to-phosphate (N-P) ratio of 15 by mixing 22 kDa linear PEI and a plasmid encoding either GFP or DCN. ECFs were generated from donor corneas as previously described. Initially, GFP was introduc...
Mobasheri A, Trujillo E, Bell S, Carter SD, Clegg PD, Martín-Vasallo P, Marples D.Chondrocytes exist in an unusual and highly variable ionic and osmotic environment in the extracellular matrix of articular cartilage. Alterations to the ionic and osmotic environment of chondrocytes influence the volume and ionic content of the cells, which, in turn, modifies the rate at which extracellular matrix macromolecules are synthesized and degraded. Thus, regulation of the water and solute content of chondrocytes will profoundly affect their anabolic and catabolic functions. The water content of cells is effectively influenced by the abundance of aquaporin (AQP) water channels. Recen...
Hellberg W, Wilson AD, Mellor P, Doherr MG, Torsteinsdottir S, Zurbriggen A, Jungi T, Marti E.Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by IgE-mediated reactions to bites of Culicoides and sometimes Simulium spp. The allergens causing IBH are probably salivary gland proteins from these insects, but they have not yet been identified. The aim of our study was to identify the number and molecular weight of salivary gland extract (SGE) proteins derived from Culicoides nubeculosus which are able to bind IgE antibodies (ab) from the sera of IBH-affected horses. Additionally, we sought to investigate the IgG subclass (IgGa, IgGb and IgGT) reactivity to thes...
Anzai T, Walker JA, Blair MB, Chambers TM, Timoney JF.To determine whether streptococcal pneumonia is caused by strains of Streptococcus zooepidemicus similar to those obtained from the tonsils of healthy horses. Methods: 5 tonsils from healthy horses, 8 tracheal washes and 6 lung specimens from foals with pneumonia, and 5 nasopharyngeal swab specimens from donkeys with acute bronchopneumonia. Methods: Variable M-like protectively immunogenic SzP proteins of 5 isolates of S. zooepidemicus from each tonsil and clinical specimen were compared, using immunoblots. The SzP gene of 13 isolates representative of various SzP immunoblot phenotypes from 1 ...
Gupta GD, Lakritz J, Kim JH, Kim DY, Kim JK, Marsh AE.Parasite-specific antibody responses to Neospora spp. and Toxoplasma gondii, antigens were detected using the indirect fluorescent antibody test (IFAT) and immunoblot analysis in a korean equine population located on Jeju island, South Korea (126 degrees 12' E and 33 degrees 34' N). For comparison, a naturally infected Neospora hughesi horse and an experimentally inoculated T. gondii equid (pony) were used. In addition, all samples were tested for antibodies to Sarcocystis neurona by immunoblot analysis. A total of 191 serum samples from clinically normal horses were evaluated. Only 2% (4 out ...
Almeida J, Ball BA, Conley AJ, Place NJ, Liu IK, Scholtz EL, Mathewson L, Stanley SD, Moeller BC.Anti-Müllerian hormone (AMH), a member of the transforming growth factor β superfamily of growth and differentiation factors, is expressed in granulosa cells of preantral and small antral ovarian follicles. In humans, AMH appeared to regulate recruitment and growth of small ovarian follicles. Furthermore, circulating AMH concentrations were elevated in women with granulosa-cell tumors (GCT). In the horse, GCTs are the most common tumor of the ovary, and a variety of endocrine assays have been used to diagnose presumptive GCTs. The objectives of the present study were to validate a heterologo...
Grimm MB, Cohen ND, Slovis NM, Mundy GD, Harrington JR, Libal MC, Takai S, Martens RJ.To determine whether mares are a clinically important source of Rhodococcus equi for their foals. Methods: 171 mares and 171 foals from a farm in Kentucky (evaluated during 2004 and 2005). Methods: At 4 time points (2 before and 2 after parturition), the total concentration of R equi and concentration of virulent R equi were determined in fecal specimens from mares by use of quantitative bacteriologic culture and a colony immunoblot technique, respectively. These concentrations for mares of foals that developed R equi-associated pneumonia and for mares with unaffected foals were compared. Data...
Montelaro RC, Robey WG, West MD, Issel CJ, Fischinger PJ.The reported serological relatedness between the major glycoproteins of human immunodeficiency virus (HIV gp120) and equine infectious anaemia virus (EIAV gp90) was examined using purified antigens in radioimmunoprecipitation (RIP), radioimmunoassay (RIA) and immunoblot assays with reference serum from acquired immunodeficiency syndrome (AIDS) patients, an anti-gp120 goat serum and EIAV-infected horse serum. To assess the contributions of glycoprotein oligosaccharide and peptide components to any observed reactivities, antigens treated with endoglycosidase F to remove carbohydrate were assayed...
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...
Wise LN, Kappmeyer LS, Silva MG, White SN, Grause JF, Knowles DP.Certain countries including the United States remain non-endemic for particular infectious diseases such as equine piroplasmosis through import restrictions and surveillance. Endemic regions often employ premunition as the primary method to control disease, however in non-endemic countries, chemosterilization combined with methods to confirm parasite elimination are required to maintain disease-free status. The ability of imidocarb diproprionate (ID) to clear persistent Theileria equi infection from infected horses has been shown through the inability of treated horses to transmit via blood tr...
Dubey JP, Venturini MC, Venturini L, McKinney J, Pecoraro M.Sera from 76 horses from Argentina were examined for antibodies to Sarcocystis neurona, Toxoplasma gondii and Neospora caninum. Antibodies to S. neurona were found in 27 (35.5%) of 76 horses using immunoblots with culture derived merozoites as antigen. Antibodies to T. gondii were found in 10 (13.1%) of 76 horses by using the modified agglutination test with formalin-fixed tachyzoites and mercaptoethanol; titers were 1:25 (two horses), 1:50 (six horses), 1:100 (two horses), and 1:200 (one horse). Antibodies to N. caninum were not found in any of the 76 horses by the use of N. caninum agglutina...
Dubey JP, Kerber CE, Granstrom DE.To determine serologic prevalence of Sarcocystis neurona, Toxoplasma gondii, and Neospora caninum in horses in Brazil. Methods: Prevalence survey. Methods: 101 Thoroughbreds in Brazil. Methods: Blood samples were obtained from horses and tested for serum antibodies against S neurona by use of an immunoblot procedure with culture-derived S neurona merozoites as antigen, and for serum antibodies against T gondii and N caninum by use of a modified agglutination test with formalin-preserved tachyzoites and mercaptoethanol. Results: Antibodies against S neurona and T gondii were detected in 36 and ...
Vardeleon D, Marsh AE, Thorne JG, Loch W, Young R, Johnson PJ.Parasite-specific antibody responses to Neospora antigens were detected using the immunofluorescent antibody test (IFAT) and immunoblot analysis in select equine populations. For comparison, a naturally infected Neospora hughesi horse and an experimentally inoculated Neospora caninum horse were used. In addition, all samples were tested for antibodies to Sarcocystis neurona by immunoblot analysis. A total of 208 samples was evaluated. The equine populations were derived from five distinct geographic regions. Locations were selected based on distribution of Didelphis virginiana, the native Nort...
Mendoza L, Nicholson V, Prescott JF.Reactions to Pythium insidiosum by sera from horses with active pythiosis were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Five strains of P. insidiosum were grown in nutrient broth and then sonicated. After centrifugation, supernatant antigens were separated by SDS-PAGE. An exoantigen of Conidiobolus coronatus was also tested. Bands with molecular weights between 97,000 and 14,000 were identified by Coomassie blue and silver staining. After being transferred to nitrocellulose, the antigens were reacted against sera from six horses w...
Khan I, Wieler LH, Melzer F, Gwida M, Santana VL, de Souza MM, Saqib M, Elschner MC, Neubauer H.The sensitivity and specificity of three commercially available complement fixation test (CFT) antigens from c.c.pro (c.c.pro), Central Veterinary Institute of Wageningen UR (CIDC) and the United States Department of Agriculture (USDA) were comparatively evaluated by testing 410 sera collected from glanders-endemic and non-endemic areas (200 true-negative randomly collected sera and 210 sera collected from experimentally immunised animals (12 rabbits, 19 horses), clinically positive (135) and culture-positive (44) horses, donkeys and mules). Immunoblotting (IB) was used as the gold standard te...
Chanter N, Talbot NC, Newton JR, Hewson D, Verheyen K.The M-protein genes of Streptococcus equi isolated from 17 outwardly healthy horses after 4 strangles outbreaks had ended, including a quarantined animal, were compared with those of S. equi isolates from 167 active cases of strangles across 4 countries. The healthy horses included 16 persistent S. equi carriers, at least one from each of the four outbreaks. These carriers, despite being outwardly healthy, had empyema of the guttural pouch(es), an enlargement of the equine Eustachian tube. A persistent carrier from two of these outbreaks, the quarantined animal and a healthy animal with normal...
Lacombe VA, Hinchcliff KW, Devor ST.To characterize insulin-sensitive glucose-transporter (GLUT-4) protein in equine tissues and determine effects of exercise and glucose administration on content of GLUT-4 protein in equine skeletal muscle. Methods: Tissue samples from 9 horses. Methods: Western blot analyses were performed on crude membrane preparations of equine tissues to characterize GLUT-4. In a crossover, randomized study, horses were strenuously exercised for 3 consecutive days and then administered 13.5% glucose or isotonic saline (0.9% NaCl; control) solution, i.v., at similar infusion rates for 12.1 hours. Samples wer...
Hagiwara K, Asakawa M, Liao L, Jiang W, Yan S, Chai J, Oku Y, Ikuta K, Ito M.To investigate the animals infected with Borna disease virus (BDV) in Xinjiang, China, we examined for BDV antibodies in the sera from groups of 20 horses, sheep and cattle, and from 165 wild rodents (18 species) by ELISA and immunoblot. The serological study disclosed the presence of antibodies to both BDV-p24 and -p40 in the horses (20%) and sheep (25%), whereas no apparent positive reaction was detected either in cattle or rodents. The results suggested that BDV is prevalent in horses and sheep in the district investigated.
Kheyar A, Martin S, St-Laurent G, Timoney PJ, McCollum WH, Archambault D.To provide a convenient and sensitive method for the detection of equine arteritis virus (EAV)-specific serum antibodies, we developed an immunoblot assay employing the EAV nucleocapsid (N) and membrane (M) proteins expressed in a procaryotic expression vector (pMAL-c2) for the production of recombinant maltose-binding (MBP) fusion proteins (MBP-N and MBP-M). The antigenic reactivity of the recombinant fusion proteins and their Xa factor cleavage EAV products was confirmed by immunoblot using horse antisera to EAV. Some horse sera, however, showed immune reactivity to the MBP fusion partner pr...
Takai S, Chaffin MK, Cohen ND, Hara M, Nakamura M, Kakuda T, Sasaki Y, Tsubaki S, Martens RJ.Rhodococcus equi isolates (462) obtained from 64 soil samples collected on 5 R. equi-endemic horse-breeding farms and isolates from 100 infected foals in Texas were examined to determine the prevalence and genotypic diversity of virulence-associated plasmids. Isolates were tested for the presence of 15-17-kDa virulence-associated protein antigens (VapA) by immunoblotting and virulence-associated plasmids by PCR. Plasmid DNAs were isolated and analyzed by digestion with restriction endonucleases for estimation of size and comparison of polymorphisims. Rhodococcus equi were isolated from soil of...
Granstrom DE, Dubey JP, Davis SW, Fayer R, Fox JC, Poonacha KB, Giles RC, Comer PF.Antigens of cultured Sarcocystis neurona merozoites were examined using immunoblot analysis. Blotted proteins were probed with S. cruzi, S. muris, and S. neurona antisera produced in rabbits, S. fayeri (pre- and post-infection) and S. neurona (pre- and post-inoculation) sera produced in horses, immune sera from 7 histologically confirmed cases of equine protozoal myeloencephalitis (EPM), and pre-suckle serum from a newborn foal. Eight proteins, 70, 24, 23.5, 22.5, 13, 11, 10.5, and 10 Kd, were detected only by S. neurona antiserum and/or immune serum from EPM-affected horses. Equine sera were ...
Avarzed A, Igarashi I, De Waal DT, Kawai S, Oomori Y, Inoue N, Maki Y, Omata Y, Saito A, Nagasawa H, Toyoda Y, Suzuki N.Monoclonal antibody (MAb) BEG3 was produced against Babesia equi parasites to define a species-specific antigen for diagnostic use. The MAb reacted with single, paired, and Maltese cross forms of B. equi, and no reaction was observed with this MAb on acetone-fixed Babesia caballi, Babesia ovata, or Babesia microti parasites in the indirect immunofluorescent antibody test. Confocal laser and immunoelectron microscopic studies showed that the antigen which was recognized by this MAb was located on the surface of B. equi parasites. This MAb recognized a 19-kDa protein of B. equi antigen and did n...
