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.
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...
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...
Liang FT, Granstrom DE, Zhao XM, Timoney JF.Sarcocystis neurona is the etiologic agent of equine protozoal myeloencephalitis (EPM). Based on an analysis of 25,000 equine serum and cerebrospinal fluid (CSF) samples, including samples from horses with neurologic signs typical of EPM or with histologically or parasitologically confirmed EPM, four major immunoblot band patterns have been identified. Twenty-three serum and CSF samples representing each of the four immunoblot patterns were selected from 220 samples from horses with neurologic signs resembling EPM and examined for inhibitory effects on the infectivity of S. neurona by an in vi...
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...
Guillot J, Sarfati J, Ribot X, Jensen HE, Latgé JP.To detect antibodies against Aspergillus fumigatus antigens in serum samples from horses and to evaluate the relevance of this method as an alternative approach to the diagnosis of mycosis of the auditory tube diverticulum (guttural pouch mycosis [GPM]). Methods: Twelve clinically normal horses (controls) and 12 horses with GPM diagnosed by endoscopic observation of characteristic mycotic plaques. Methods: Antibodies to A fumigatus antigens were detected in serum by use of an ELISA and immunoblot analysis with extracellular antigens. Results: Antibodies against A fumigatus antigens were found ...
Lipscomb DL, Boudreaux MK, Paxton R, Spano J, Welles EG, Schumacher J.To establish the existence of platelet-derived proteins in equine plasma, with the future goal of developing an assay for the detection of in vivo platelet activation. Methods: 5 mature healthy horses. Methods: Platelet-rich plasma and platelet-poor plasma were prepared from anticoagulated blood. Platelets were separated from plasma proteins by gel filtration, then activated with 0.5 microM platelet-activating factor. Protease inhibitors were added, and the released platelet proteins were harvested. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on the released platele...
Liang FT, Granstrom DE, Timoney JF, Shi YF.We report a simple, economical, and efficient protocol for protein purification from cells. First, proteins of cell lysates were separated by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted to protein-blotting membrane. The blots were stained with Coomassie blue or developed by immunoblotting to visualize specific proteins. The bands corresponding to those visible by immunoblotting were excised from the dye-stained blots and subjected to isoelectric focusing. The focused gel was stained with Coomassie blue. Finally, the stained bands were excise...
Fontanals AM, Becú T, Polledo G, Gaskin CK, Braun M.An R. equi vaccine, prepared under conditions which induce the expression of many antigens, and which has given encouraging results in field trials, was analyzed by SDS-PAGE and immunoblots and compared with other R. equi preparations: a preparation made in with the same technique from a nonvirulent isolate (virulence associated protein negative, VapA-negative); a whole cell preparation of a VapA-positive R. equi, prepared as a standard bacterin; and a semipurified VapA preparation (APTX). The antigens in these preparations were analyzed using hyperimmune sera (from adult horses vaccinated wit...
Martínez-Torrecuadrada JL, Díaz-Laviada M, Roy P, Sánchez C, Vela C, Sánchez-Vizcaíno JM, Casal JI.Fifteen horses were experimentally infected with African horse sickness virus (AHSV) serotype 4. To learn more about the time course of production and specificity of AHSV-specific antibodies, sera were analyzed by immunoblot analysis. Only animals that survived for more than 9 days were able to develop a humoral immune response detectable by immunoblotting. The earliest serological markers corresponded mainly to VP5, VP6, and NS2 and to a lesser extent to VP3, NS1, and NS3. Neutralizing antibodies to VP2 were not detected by immunoblotting, suggesting that they are mostly conformation dependen...
Johnsen , Thanh DB, Ly Q, Smestad Paulsen B, Wold JK.IgE-binding components in an extract of horse dander were analyzed, especially with regard to the glycoprotein allergens. After SDS-PAGE under reducing conditions and blotting, several of the glycoprotein IgE-binding components, including two distinct bands of 27 and 31 kDa, were detected. Together with several other bands, they were shown to bind to the lectins Sambucus nigra agglutinin (SNA) and Datura stramonium agglutinin (DSA), indicating terminal sialic acid linked alpha 2 --> 6 to galactose, and galactose linked beta 1 --> 4 to N-acetylglucosamine, respectively. Carbohydrate analy...
Felix K, Ferrándiz R, Einarsson R, Dreborg S.Some patients who are allergic to horses have reported that they can tolerate certain breeds, and the presence of breed-specific allergens has been suggested. Breeders and patients with asthma have claimed that Bashkir horses are nonallergenic. We used sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting to determine IgE-binding profiles of extracts of dander obtained from horses of several breeds. We found considerable inter-breed and within-breed variation but no breed-specific allergens. Danders from all breeds investigated contained the most important allergens, and ...
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,...
Bell K.Transferrin, albumin, 6-phosphogluconate dehydrogenase and vitamin D-binding protein polymorphisms were detected in 242 feral and domesticated Australian donkeys by polyacrylamide gel electrophoresis, starch gel electrophoresis, autoradiography, immunoblotting with specific antisera and activity staining. All four TF and two ALB variants were donkey specific while only one of the PGD variants was donkey specific. The two GC variants were electrophoretically identical to the Equus caballus F and S proteins. Available evidence suggested that the TF, ALB, PGD and GC systems are controlled by co-d...
Sugiura T, Nakamura H.The zinc content in platelets from rabbits, humans and horses was determined, and the levels of zinc were found to be significantly higher (3 micrograms/10(10) cells) than those in other peripheral blood cells. About 70% of the zinc in the supernatants of platelet lysates could be detected. From the results of gel filtration analysis, the zinc in platelet lysates was found to be bound with a low-molecular-weight protein (MW 6,000-8,000) detected as metallothionein (MT) on the basis of antigenic properties determined by enzyme-linked immunoassay and immunoblotting analysis using monoclonal anti...
Fjeldsgaard BE, Paulsen BS.Extracts of horse dander (HD) and horse hair and skin scrapings (HHSS) have been compared with respect to their content of proteins and carbohydrates. The protein content of HD is more than double that of HHSS, while the carbohydrate content is of the same order. SDS-PAGE and IEF, both combined with immunoblotting, and CIE/CRIE showed the IgE-binding ability of the proteins/glycoproteins present in the two extracts. SDS-PAGE/immunoblotting showed the presence of mainly the same IgE-binding bands in the two extracts. Nine were detected in HD, and seven in HHSS. Four of these were glycoproteins....
DeVico AL, Issel CJ, Le Grice SF, Payne SL, Montelaro RC, Sarngadharan MG.The immunogenicity of the equine infectious anemia virus (EIAV) reverse transcriptase (RT) was examined by immunoblot assay with recombinant EIAV RT. All of the 19 sera from EIAV-infected horses tested contained antibodies that recognized EIAV RT and directly inhibited the polymerase activity of the enzyme. An examination of sera obtained sequentially from two experimentally infected animals revealed that anti-RT antibodies arise early in infection and increase in level. The appearance of the antibodies correlated with progression toward the asymptomatic period of infection.
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 ...
Miller CC, Fayrer-Hosken RA, Timmons TM, Lee VH, Caudle AB, Dunbar BS.This study was designed to explore the composition of the equine zona pellucida (EZP) by one- and two-dimensional polyacrylamide gel electrophoresis (1D- and 2D-PAGE), silver staining and immunoblotting techniques. Antral follicles palpable on frozen-thawed equine ovaries were aspirated with a needle and syringe, and the resultant follicular fluid, cellular material and oocytes were pooled. Oocytes were placed in Petri dishes, moved by narrow-bore pipette to droplets of phosphate-buffered saline (PBS) and mechanically cleaned of cumulus cells. The EZP from these collected oocytes was solubiliz...
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...
Parma AE, Cerone SI, Sansinanea SA.The antigenic relationship between Leptospira interrogans, equine cornea and lens was previously noted in our studies. Serum antibodies from horses inoculated with serovars wolffi, pomona, icterohaemorrhagiae, and tarassovi, were able to bind to five antigenic fractions from both cornea and lens, as demonstrated by immunoblotting. These antigens seem to be made up of protein and carbohydrates. After treatment with periodate for cleavage of glycoside ring structures, those fractions kept their condition of target for anti-Leptospira antibodies. Nevertheless, all fractions lost that condition af...
Divers TJ, Timoney JF, Lewis RM, Smith CA.A 12-year-old thoroughbred gelding died from diffuse global glomerulonephritis, 3 months after a lower respiratory infection from which Streptococcus zooepidemicus was isolated. Immunopathological studies (immunofluorescence, immunodiffusion, immunoperoxidase testing and immunoblotting) indicated the presence of an immune reactant renal disease associated with IgG antibody and streptococcal antigens.
Schuberth HJ, Anders I, Pape U, Leibold W.The cells of 60 randomly selected Hannoveranian warm-blooded horses were subjected to one-dimensional isoelectric focusing and immunoblotting with a cross-reacting monoclonal antibody (Bo 1) recognizing bovine class I antigens. The banding patterns were correlated with the serologically defined specificities of the ELA-A locus. ELA-A2 was correlated with four bands, while ELA-A5, ELA-W18, ELA-A6, ELA-A14 and ELA-A9 were correlated with a single band each. The complexity of the pattern and additional polymorphic bands which could not be correlated to any of the known ELA specificities may indic...
Barakat-Walter I, Deloulme JC, Sensenbrenner M, Labourdette G.We have previously shown that neuroblasts from cerebral hemispheres of 6-day-old chick embryos are able to proliferate when grown in the presence of fetal calf serum. We report here that in the presence of horse serum alone the proliferative rate of neuroblasts is strongly reduced. A high proliferative rate is restored upon the addition of bovine transferrin and to a lesser extent with added FeSO4 or hemin. These findings suggest that the transferrin of horse serum cannot be used by chick neuroblasts in vitro, while bovine transferrin exogenously added is active in promoting cell proliferation...
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...
Patterson SD, Bell K, Shaw DC.1. The donkey postalbumin protein has been shown to be the equivalent of human alpha 1 B-glycoprotein by protein immunoblotting and N-terminal amino acid sequence. 2. The horse A1B system (already identified as the homologue of human alpha 1 B-glycoprotein) and the donkey alpha 1 B-glycoprotein were characterized further for terminal sialic acid content, isoelectric point, amino acid composition and affinity for the dye-ligand, Cibacron Blue F3GA (known to bind human alpha 1 B-glycoprotein). 3. Two new alleles in the horse A1B system were found, bringing the total number of alleles to five. No...
Groschup M, Müller HP, Weiss R, Schliesser T.For the determination of a species-specific antigen of Streptococcus (S.) equi, acid extracts of group C streptococcal strains from horses (S. equi, S. zooepidemicus, S. equisimilis) were investigated using polyacrylamide gel electrophoresis and the immunoblotting technique. Using sera of horses suffering from strangles as well as sera from horses with respiratory infection of unknown etiology, Western blotting yielded more or less multiple banding reactions with bands in the 70, 54, 42, 40, and 31-28 kd molecular weight ranges against extracts of all of the 3 different bacterial species. Howe...
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...
Patterson SD, Bell K.An affinity method was developed to investigate the interaction between protease and protease inhibitor by incorporating a protease incubation step into a two-dimensional electrophoretic separation of the plasma protease inhibitory proteins. This involved the application of the isoelectric focusing gel to filter paper saturated in the protease of choice before being placed on the second-dimensional polyacrylamide electrophoresis gel. General protein staining or immunoblotting was used to detect the protein or ligand in the complex. An in situ oxidation method was developed using the reagent ch...
Juneja RK, Gahne B, Lukka M, Ehnholm C.By using immunoblotting with antiserum specific to human plasma apolipoprotein A-IV (apoA-IV), a previously reported polymorphic plasma protein of dogs viz postalbumin-2 (Pa2) and one of horses viz serum protein 2 (SP2), were identified as apoA-IV of these species. This along with earlier published results implied that: (1) both dog and horse show a high degree of polymorphism at the APOA4 locus with three common alleles in each of the two species; and (2) apoA-IV phenotyping in these two species can be done by analysing plasma/serum samples by a simple method of two-dimensional electrophoresi...
van Andel AC, Gruys E, Kroneman J, Veerkamp J.Out of approximately 16,000 horses referred for clinical examination, nine had amyloidosis. Six of these horses had localised amyloid deposits in the wall of the nasal meatus and ventral turbinates associated with epistaxis. Horse 1 also developed malignant histiolymphocytic lymphosarcomas. The amyloid deposits were potassium permanganate-resistant and tryptophan-positive. Gel filtration of solubilised amyloid fibrils from Horse 1 revealed a major retarded fraction with an apparent molecular weight of 20 kD. This protein had an amino acid composition similar to human AL-amyloid proteins and ho...
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...
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...
Liang FT, Granstrom DE, Timoney JF, Shi YF.We report a simple, economical, and efficient protocol for protein purification from cells. First, proteins of cell lysates were separated by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted to protein-blotting membrane. The blots were stained with Coomassie blue or developed by immunoblotting to visualize specific proteins. The bands corresponding to those visible by immunoblotting were excised from the dye-stained blots and subjected to isoelectric focusing. The focused gel was stained with Coomassie blue. Finally, the stained bands were excise...
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...
Johnsen , Thanh DB, Ly Q, Smestad Paulsen B, Wold JK.IgE-binding components in an extract of horse dander were analyzed, especially with regard to the glycoprotein allergens. After SDS-PAGE under reducing conditions and blotting, several of the glycoprotein IgE-binding components, including two distinct bands of 27 and 31 kDa, were detected. Together with several other bands, they were shown to bind to the lectins Sambucus nigra agglutinin (SNA) and Datura stramonium agglutinin (DSA), indicating terminal sialic acid linked alpha 2 --> 6 to galactose, and galactose linked beta 1 --> 4 to N-acetylglucosamine, respectively. Carbohydrate analy...
Fontanals AM, Becú T, Polledo G, Gaskin CK, Braun M.An R. equi vaccine, prepared under conditions which induce the expression of many antigens, and which has given encouraging results in field trials, was analyzed by SDS-PAGE and immunoblots and compared with other R. equi preparations: a preparation made in with the same technique from a nonvirulent isolate (virulence associated protein negative, VapA-negative); a whole cell preparation of a VapA-positive R. equi, prepared as a standard bacterin; and a semipurified VapA preparation (APTX). The antigens in these preparations were analyzed using hyperimmune sera (from adult horses vaccinated wit...
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...
Patterson SD, Bell K, Shaw DC.1. The donkey postalbumin protein has been shown to be the equivalent of human alpha 1 B-glycoprotein by protein immunoblotting and N-terminal amino acid sequence. 2. The horse A1B system (already identified as the homologue of human alpha 1 B-glycoprotein) and the donkey alpha 1 B-glycoprotein were characterized further for terminal sialic acid content, isoelectric point, amino acid composition and affinity for the dye-ligand, Cibacron Blue F3GA (known to bind human alpha 1 B-glycoprotein). 3. Two new alleles in the horse A1B system were found, bringing the total number of alleles to five. No...
Miller CC, Fayrer-Hosken RA, Timmons TM, Lee VH, Caudle AB, Dunbar BS.This study was designed to explore the composition of the equine zona pellucida (EZP) by one- and two-dimensional polyacrylamide gel electrophoresis (1D- and 2D-PAGE), silver staining and immunoblotting techniques. Antral follicles palpable on frozen-thawed equine ovaries were aspirated with a needle and syringe, and the resultant follicular fluid, cellular material and oocytes were pooled. Oocytes were placed in Petri dishes, moved by narrow-bore pipette to droplets of phosphate-buffered saline (PBS) and mechanically cleaned of cumulus cells. The EZP from these collected oocytes was solubiliz...
Schuberth HJ, Anders I, Pape U, Leibold W.The cells of 60 randomly selected Hannoveranian warm-blooded horses were subjected to one-dimensional isoelectric focusing and immunoblotting with a cross-reacting monoclonal antibody (Bo 1) recognizing bovine class I antigens. The banding patterns were correlated with the serologically defined specificities of the ELA-A locus. ELA-A2 was correlated with four bands, while ELA-A5, ELA-W18, ELA-A6, ELA-A14 and ELA-A9 were correlated with a single band each. The complexity of the pattern and additional polymorphic bands which could not be correlated to any of the known ELA specificities may indic...
Magnarelli LA, Van Andel AE, Ijdo JW, Heimer R, Fikrig E.To diagnose granulocytic ehrlichiosis in horses, compare results of indirect fluorescent antibody (IFA) staining procedures with those of immunoblot analysis, and compare serologic test findings with polymerase chain reaction (PCR) results. Methods: 69 horses with high rectal temperatures (> or = 39 C) and lethargy, anorexia, or limb edema. Methods: 43 convalescent serum samples obtained from 38 horses 2 to 18 weeks after onset of illness were analyzed by use of immunoblot procedures and IFA staining methods, using the NCH-1 or BDS ehrlichial strains. Blood samples from 69 acutely ill horse...
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...
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...
The Journal of parasitologyNovember 26, 2004
Volume 90, Issue 5 1027-1033 doi: 10.1645/0022-3395(2004)090[1027:AEOARB]2.0.CO;2
Gupta GD, Lakritz J, Saville WJ, Livingston RS, Dubey JP, Middleton JR, Marsh AE.Sarcocystis neurona is the primary parasite associated with equine protozoal myeloencephalitis (EPM). This is a commonly diagnosed neurological disorder in the Americas that infects the central nervous system of horses. Current serologic assays utilize culture-derived parasites as antigen. This method requires large numbers of parasites to be grown in culture, which is labor intensive and time consuming. Also, a culture-derived whole-parasite preparation contains conserved antigens that could cross-react with antibodies against other Sarcocystis species and members of Sarcocystidae such as Neo...
DeVico AL, Issel CJ, Le Grice SF, Payne SL, Montelaro RC, Sarngadharan MG.The immunogenicity of the equine infectious anemia virus (EIAV) reverse transcriptase (RT) was examined by immunoblot assay with recombinant EIAV RT. All of the 19 sera from EIAV-infected horses tested contained antibodies that recognized EIAV RT and directly inhibited the polymerase activity of the enzyme. An examination of sera obtained sequentially from two experimentally infected animals revealed that anti-RT antibodies arise early in infection and increase in level. The appearance of the antibodies correlated with progression toward the asymptomatic period of infection.
Bell K.Transferrin, albumin, 6-phosphogluconate dehydrogenase and vitamin D-binding protein polymorphisms were detected in 242 feral and domesticated Australian donkeys by polyacrylamide gel electrophoresis, starch gel electrophoresis, autoradiography, immunoblotting with specific antisera and activity staining. All four TF and two ALB variants were donkey specific while only one of the PGD variants was donkey specific. The two GC variants were electrophoretically identical to the Equus caballus F and S proteins. Available evidence suggested that the TF, ALB, PGD and GC systems are controlled by co-d...
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...
Timoney JF.An R antigen of the group C streptococcus S. equi that cross reacts with a similar antigen of S. zooepidemicus has been identified and characterized. It is acid, heat and trypsin resistant, but pepsin sensitive and has an isoelectric point of 4.8. The amino acids in highest concentration are glutamic, aspartic, alanine, leucine, and valine. Bacterial components released in a French Press contain large amounts of R antigen, which is present also in culture supernatants and acid extracts. It has a molecular weight of about 82,000. Trypsin extraction of cells yields molecules of predominantly 56,...
Groschup M, Müller HP, Weiss R, Schliesser T.For the determination of a species-specific antigen of Streptococcus (S.) equi, acid extracts of group C streptococcal strains from horses (S. equi, S. zooepidemicus, S. equisimilis) were investigated using polyacrylamide gel electrophoresis and the immunoblotting technique. Using sera of horses suffering from strangles as well as sera from horses with respiratory infection of unknown etiology, Western blotting yielded more or less multiple banding reactions with bands in the 70, 54, 42, 40, and 31-28 kd molecular weight ranges against extracts of all of the 3 different bacterial species. Howe...
Mobarak MS, Ryan MF.Adult Strongylus vulgaris, collected from the caecum of infected horses and embedded in paraplast using standard methods, were sectioned for immunohistochemistry (IHC) studies. Antibodies were raised in rabbit against the excretory-secretory product (ESP) and against two constituent protein bands (28-30 kDa). The use of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA) and immunoblotting indicated the immunogenicity of ESP and of the subunits (28-30 kDa). In ELISA, both rabbit hyperimmune sera recognized the ESP and (28-30 kDa) ban...
Lipscomb DL, Boudreaux MK, Paxton R, Spano J, Welles EG, Schumacher J.To establish the existence of platelet-derived proteins in equine plasma, with the future goal of developing an assay for the detection of in vivo platelet activation. Methods: 5 mature healthy horses. Methods: Platelet-rich plasma and platelet-poor plasma were prepared from anticoagulated blood. Platelets were separated from plasma proteins by gel filtration, then activated with 0.5 microM platelet-activating factor. Protease inhibitors were added, and the released platelet proteins were harvested. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on the released platele...
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...
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...
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 ...
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...
Finley MR, Li Y, Hua F, Lillich J, Mitchell KE, Ganta S, Gilmour RF, Freeman LC.In dogs and in humans, potassium channels formed by ether-a-go-go-related gene 1 protein ERG1 (KCNH2) and KCNQ1 alpha-subunits, in association with KCNE beta-subunits, play a role in normal repolarization and may contribute to abnormal repolarization associated with long QT syndrome (LQTS). The molecular basis of repolarization in horse heart is unknown, although horses exhibit common cardiac arrhythmias and may receive drugs that induce LQTS. In horse heart, we have used immunoblotting and immunostaining to demonstrate the expression of ERG1, KCNQ1, KCNE1, and KCNE3 proteins and RT-PCR to det...
Parma AE, Cerone SI, Sansinanea SA.The antigenic relationship between Leptospira interrogans, equine cornea and lens was previously noted in our studies. Serum antibodies from horses inoculated with serovars wolffi, pomona, icterohaemorrhagiae, and tarassovi, were able to bind to five antigenic fractions from both cornea and lens, as demonstrated by immunoblotting. These antigens seem to be made up of protein and carbohydrates. After treatment with periodate for cleavage of glycoside ring structures, those fractions kept their condition of target for anti-Leptospira antibodies. Nevertheless, all fractions lost that condition af...
Csellner H, Walker C, Love DN, Whalley JM.An equine herpesvirus 1 (EHV-1) mutant was constructed by inserting a lacZ expression cassette into the intergenic region upstream of gene 62 (glycoprotein L; gL) and downstream of gene 63 (a homologue of the herpes simplex virus transcriptional activator ICP0). The recombinant lacZ62/63-EHV-1 had similar growth kinetics in cell culture to those of the parental wild type (wt) virus, with indistinguishable cytopathic effects and plaque morphology. Reverse transcriptase PCR confirmed that the lacZ insertion did not interfere with transcription of gL and immunoblot analysis indicated there was no...
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...
