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.
Kao M, Hamir AN, Rupprecht CE, Fu ZF, Shankar V, Koprowski H, Dietzschold B.Sera from 295 horses in the USA were examined by an indirect immunofluorescence assay and Western blot assays to determine the prevalence of Borna disease virus infection. Eight (2.7 per cent) of the samples were positive in both assays, and 18 (6.1 per cent) were positive only in the Western blot assay. The indirect fluorescence titres ranged from 1:20 to 1:80 of antibodies recognising the virus-specific antigen from Borna disease virus-infected cells. The purified virus-specific proteins isolated from infected rat brains were recognised by positive equine serum samples after immunostaining b...
Laviada MD, Arias M, Sánchez-Vizcaíno JM.The structural and non-structural proteins induced by African horsesickness virus serotype 4 (AHSV-4) in infected Vero cells were analysed by SDS-PAGE. Twenty-two virus-induced polypeptides were detected in infected cells by comparison with the polypeptides of mock-infected cells, of which four major (VP2, VP3, VP5 and VP7) and three minor (VP1, VP4 and VP6) structural proteins and four non-structural proteins (P58, P48, P21 and P20) were shown to be virus-coded, as deduced from electrophoretic and antigenic studies of purified virions and infected cells. The proteins that elicit the major ant...
Bürki F, Rossmanith W, Rossmanith E.Serological diagnosis of equine infectious anemia is of necessity group-reactive, i.e. based on viral core protein p26, because viral envelope components as well as the host's immune response to them undergo rapid antigenic change. Since 1970 the agar gel-immunodiffusion test ("Coggins-test") has been the diagnostic method of choice. Recently, ELISA tests have been introduced for faster and theoretically more sensitive serodiagnosis, while Western blots have been used to clarify doubtful results obtained in Coggins-tests. A commercial competitive ELISA was found to give practically equivalent ...
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...
Cohen ND, Heck FC, Heim B, Flad DM, Bosler EM, Cohen D.Four hundred sixty-nine serum samples were obtained from horses admitted to the internal medicine service of the Texas Veterinary Medical Center between Jan 1 and Dec 31, 1990. Serum samples were tested by ELISA for antibody to Borrelia burgdorferi. Of these 469 samples, 1 (0.2%) was repeatedly seropositive for the organism by ELISA. Confirmatory testing by protein immunoblot was negative. The observed seroprevalence was 0%; the upper bound of the 95% confidence interval was 0.6%. These findings indicate the evidence of infection with B burgdorferi is presently uncommon in horses in central Te...
Böse R, Daemen K.Babesia caballi-infected or normal equine erythrocytes were solubilized in sodium dodecyl sulfate (SDS) buffer and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Antigens were allowed to react with sera from horses experimentally or field-infected with B. caballi and with sera from non-infected horses. Major babesial antigens recognized by immune sera had apparent mol. wts of 141, 112, 70, 50, 48, 34, and 30 kDa. The polypeptides at 50 and 48 kDa were recognized earliest and throughout infection, but also weakly by 3/100 equine sera tested negative and 1/33...
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...
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...
Stewart F, Tuffnell PP.A 514 bp cDNA transcript coding for 78% of horse (Equus caballus.) GH has been cloned and sequenced. The deduced amino acid sequence corresponded precisely to that previously obtained by protein sequencing and, in addition, provided new sequence information for the signal peptide. The missing 3' fragment of the cDNA was reconstructed using synthetic oligonucleotides and site-specific directed mutagenesis. The complete cDNA sequence was then inserted into an expression vector (PIN-III-lppp-5) which utilizes a bacterial signal peptide to secrete the expressed product into the periplasmic space o...
Chong YH, Ball JM, Issel CJ, Montelaro RC, Rushlow KE.Defined segments of the transmembrane envelope glycoprotein (gp45) of equine infectious anemia virus were expressed as TrpLE fusion proteins and examined for their reactivity in Western immunoblots against a diverse panel of equine immune sera. The most immunogenic region of gp45 was localized to its amino terminus, positioned between the hydrophobic fusion and the transmembrane domains. A series of overlapping synthetic peptides were used in enzyme-linked immunosorbent assays to define an immunodominant epitope within this region. In contrast, the carboxy-terminal half of gp45 displayed both ...
Piquette GN, Kenney RM, Sertich PL, Yamoto M, Hsueh AJ.The association of equine granulosa-theca cell tumors with atrophied contralateral ovaries and abnormal estrous cycles suggests that these tumors produce hormones that affect pituitary gonadotropin production. Because inhibin, a heterodimer protein secreted by granulosa cells, decreases FSH production, we examined the presence of inhibin alpha- and beta A-subunits and their mRNAs in ovarian tumors obtained from three mares. These tumors contained neoplastic cords and nodules, multiple fluid-filled cysts, and a predominance of neoplastic granulosa cells. Reduced proteins from tumor-conditioned ...
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...
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...
Egberink HF, Ederveen J, Montelaro RC, Pedersen NC, Horzinek MC, Koolen MJ.Feline immunodeficiency virus (FIV) grown in cat lymphocyte and thymocyte cultures was labelled with L-[35S]methionine or [3H]glucosamine and virus-coded proteins were identified using immunoprecipitation. Polypeptides with apparent Mr values of 15K, 24K, 43K, 50K, 120K and 160K were detected. An additional polypeptide of 10K was detected by Western blot analysis. The two highest Mr species sometimes appeared as one band, of which only the 120K polypeptide was glycosylated. In the presence of tunicamycin gp120 was no longer detectable and a non-glycosylated precursor of 75K was found instead. ...
Milne EM.Equine serum haptoglobin was separated by polyacrylamide gel isoelectric focusing and visualized by protein staining or Western blotting. Conventional protein staining revealed up to three bands in the pI range 4.17 to 4.44. The blotting technique, however, showed an anodal group of 8 to 10 bands with a pI range of 4.11 to 4.52 and a cathodal group of 4 to 6 bands with a range of 4.55 to 5.14. The blotting method revealed that equine haptoglobin migrates outside the prealbumin area, in contrast to previous reports.
Patterson SD, Bell K.The carbohydrate side chains of the major plasma serpins of the horse and wallaby have been characterized by lectin analyses of protein blots from two-dimensional gels using the major human plasma serpin, alpha 1-protease inhibitor, as a control. Eight lectins were used in the characterization in conjunction with enzymatic deglycosylation of complex and high mannose side chains, chemical desialylation and defucosylation, and one round of 'Smith degradation', all being performed on the nitrocellulose blots. Assuming a standard complex side chain structure, the results of the 21 lectin/treatment...
Welsch U, Schumacher U, Buchheim W, Schinko I, Jenness P, Patton S.A specific secretory product of the lactating mammary gland are triglyceride fat globules which are enveloped by a very complex membrane, the milk fat globule membrane (MFGM). In different mammalian species (man, rhesus monkey, horse, goat, sheep, cow, grey seal, camel, alpaka) the glycoproteins of this membrane have been analyzed by gel electrophoresis, Western blotting and lectin histochemistry. A remarkable intra- and interspecific variability of these glycoproteins has been detected pointing to so far unknown physiological adaptions, which may play a role in the intestine of the new born. ...
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...
Dutta SK, Mattingly BL, Shankarappa B.The antibody response and the antibody reactivity to component antigens of Ehrlichia risticii were studied in horses with induced Potomac horse fever. These horses had no detectable antibodies to E. risticii in their preinoculation (PrI) sera by indirect fluorescent-antibody assay and enzyme-linked immunosorbent assay (ELISA). All the horses exhibited typical disease features following experimental infection and responded with specific antibodies, as measured by ELISA and indirect fluorescent-antibody assay. A primary antibody response was detected in 70% of the horses, while a secondary-type ...
Payne SL, Rushlow K, Dhruva BR, Issel CJ, Montelaro RC.Previous characterizations of equine infectious anemia virus (EIAV) glycoprotein variation by DNA sequence analysis and epitope mapping using monoclonal antibodies (MAbs) have revealed the presence of conserved and variable regions within the EIAV env gene. To extend these studies, fragments of the EIAV envelope proteins gp90 and gp45 were expressed in Escherichia coli and used in Western blot analysis with a diverse panel of equine immune sera to identify antigenic segments. All sera from EIAV-infected animals reacted with the carboxyl terminal portion of gp90 and the amino terminal portion o...
Meredith DM, Stocks JM, Whittaker GR, Halliburton IW, Snowden BW, Killington RA.Equine herpesvirus types 1 and 4 (EHV-1 and EHV-4) labelled with [14C]glucosamine were purified from infected cell culture medium and profiles of their structural proteins were obtained that enabled identification of the major glycoproteins. Nine glycosylated polypeptides were identified for each virus. Preparations of the purified viruses each contained a glycoprotein which was linked by disulphide bonds, as determined by diagonal gel electrophoresis under reducing/non-reducing conditions. High Mr forms of this glycoprotein were detected for EHV-1 when the sample was not heated. The EHV-1 pro...
Rossmanith W, Horvath E.After electrophoretic separation in SDS-PAGE structural proteins of the virus of Equine Infectious Anemia (EIA) were easily blotted by the semi-dry-blotting method onto nitrocellulose filters. Strips of these filters were used for antibody demonstration, and positive reactions thereof were intensified by a biotin-avidin-peroxidase system. Sensitivity of this system was so high as to allow readable interpretation of bands up to the dilution of 1:6,400 of a strongly positive serum. Frequently this procedure allowed to make a firm diagnostic Western-Blot diagnosis on far weaker equine sera. Inter...
Matsushita T, Hesterberg LK, Porter JP, Smith BJ, Newman LE.Two diagnostic tests are approved for detecting antibody to equine infectious anemia virus: the agar-gel immunodiffusion (AGID) test and the competitive enzyme-linked immunosorbent assay (ELISA). A total of 420 sera from National Veterinary Services Laboratories check sets were tested with the AGID and competitive ELISA. A 100% correlation was obtained. The AGID and competitive ELISA were further used to test difficult samples with low levels of equine infectious anemia antibody (weak positives). A third test (Western blot) was also used with these weak positive samples to resolve any discorda...
Rikihisa Y, Pretzman CI, Johnson GC, Reed SM, Yamamoto S, Andrews F.Ehrlichia risticii has a close antigenic relationship to E. sennetsu. Sera of ponies experimentally infected with E. risticii, the etiologic agent of Potomac horse fever, consistently reacted with E. sennetsu, a human pathogen, in indirect fluorescent-antibody (IFA) testing, while human E. sennetsu convalescent serum reacted with E. risticii by IFA testing and immunoferritin labeling of cells infected in vitro. Two ponies injected intravenously with live E. sennetsu did no develop clinical illness. Subsequent injection with live E. sennetsu did not develop clinical illness. Subsequent injectio...
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...
Bridges CG, Ledger N, Edington N.Ponies, without evidence of previous exposure to Equine herpes virus-1 (EHV-1), were experimentally infected with EHV-1 subtype 2 and investigated for lymphocyte transformation to virus-infected cell polypeptides, as shown by separation with gel electrophoresis. Animals made significant responses to Western blot fractions that corresponded to molecular weights of approximately 30,000, 40,000-45,000, 60,000-65,000, 80,000-95,000 and 100,000-140,000 MW. These molecular weight ranges correlated with the positions of major EHV-1 subtype 2 glycoproteins that were found at migration distances approx...
Nishita T, Matsushita H.1. Carbonic anhydrase III (CA-III) from different mammalian species (horse, cow, dog, cat, rat and rabbit) has been analyzed by the immunodiffusion technique with anti-equine CA-III serum. 2. Immunodiffusion demonstrated the absence of cross-reactivity between isozyme CA-I, CA-II, and CA-III. 3. Cross-reactions were observed between the CA-III from all the species examined except the rabbit. 4. Molecular weights and isoelectric points of CA-III from different species were determined by Western blotting.
Bosler EM, Cohen DP, Schulze TL, Olsen C, Bernard W, Lissman B.By using paired sera the IgM and IgG host responses were analyzed in dogs with ELISA and Western blot techniques. Antibodies in clinical seropositive dogs bound to 4-25 IgM and up to 40 or more IgG antigenic determinants. Early IgM response to the 41-kDa flagellin persisted for at least 9 months and involved as many as seven other peptides. IgG response expanded later in the disease and involved more immunogens than are currently recognized in late human disease. A percentage of asymptomatic dogs that later developed clinical symptoms were seropositive. Immunoblot studies suggested that B. bur...
Hussain KA, Issel CJ, Schnorr KL, Rwambo PM, West M, Montelaro RC.Monoclonal antibodies (MCAbs) were used to dissect the antigenic sites of the surface glycoproteins of the prototype cell-adapted Wyoming strain of equine infectious anemia virus (EIAV). Serologic reactivities of these MCAbs were determined by ELISA, additive ELISA, competitive ELISA, and Western blot assays. The results indicated that antigenic reactivity of gp90 was localized on at least four distinct epitopes, two of which were important in neutralization. Our studies also revealed that these epitopes were localized on overlapping antigenic sites on gp90. On the other hand, only two distinc...
Welsch U, Buchheim W, Schumacher U, Schinko I, Patton S.Horse milk fat globules (MFGs) and casein micelles were studied using freeze fracturing, freeze etching and thin-section electron microscopy, as well as lectin histochemistry, gel electrophoresis, and Western blotting. Horse MFGs were found to be relatively small, their average volume-surface diameter being about 2.75 microns. The MFG membrane is composed of three layers: an inner proteinaceous coat occasionally having a paracrystalline substructure, a unit membrane, and a prominent filamentous glycocalyx. The last is rich in glycoconjugates, as revealed by its binding of various lectins. In a...
Kearns CF, Lenhart JA, McKeever KH.Erythropoietin (EPO) is the primary hormone of erythropoiesis. Administration of recombinant human erythropoietin (rhuEPO) to improve racing performance in the horse represents a new form of blood doping, which has been associated with increased mortality. While immunoassay kits have become plentiful, very few commercial hormone assays are made specifically for equine research. There is a strong degree of sequence homology reported for EPO among species, which has allowed antibodies designed for human EPO research to be used to determine EPO concentration in other species. The objective of the...
Chen J, Guo X, Li L.The nucleocapsid (N) protein is the most conserved structural protein in equine arteritis virus (EAV). This study aimed to identify the minimal conserved B cell epitope on the EAV N protein. The purified N protein was used to immunize mice for preparing monoclonal antibody (mAb). The reactivity of mAb was evaluated by Western blot and immunofluorescence assay. Moreover, 11 overlapping peptides (named MBP-N1 to MBP-N11) were designed to localize the linear antigenic epitope within the N protein. The peptides were identified by indirect enzyme-linked immunosorbent assay (ELISA) and Western blot....
Wilson AD, Harwood L, Torsteinsdottir S, Marti E.Immunoglobulin E forms a minor component of serum antibody in mammals. In tissues IgE is bound by FcvarepsilonRI receptors on the surface of mast cells and mediates their release of inflammatory substances in response to antigen. IgE and mast cells have a central role in immunity to parasites and the pathogenesis of allergic diseases in horses and other mammals. This paper describes the production of several novel monoclonal antibodies that detect native equine IgE in immunohistology, ELISA and Western blotting. An antigen capture ELISA to quantify equine IgE in serum has been developed using ...
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...
Deschene K, Céleste C, Boerboom D, Theoret CL.As a transient hypoxic state exists within skin wounds in horses and may be important for the healing process, this study sought to identify a molecular hypoxia response occurring in horse limb and body wounds healing by second intention. Hypoxia-inducible factor 1α (HIF1α) protein expression was studied throughout repair by Western blotting and immunofluorescence. Paradoxically, HIF1α was strongly expressed in intact skin and its expression decreased dramatically following wounding (p<0.01), despite the expected hypoxic state within the wounded tissue. HIF1α levels reincreased in parallel...
Jeronimo C, Archambault D.Equine arteritis virus (EAV), an enveloped positive-stranded RNA virus, is the prototype of the arterivirus group. In a previous paper (A. Kheyar, S. Martin, G. St.-Laurent, P. J. Timoney, W. H. McCollum, and D. Archambault, Clin. Diagn. Lab. Immunol. 4:648-652, 1997), we have shown that the unglycosylated membrane (M) protein, which is composed of 162 amino acids (aa), is a major target of equine antibody to EAV. In order to determine the antigenic regions of the M protein, the cDNA encoding the M protein of EAV was inserted into the procaryotic expression vector pGEX-4T-1 to produce recombin...
Hussain KA, Issel CJ, Schnorr KL, Rwambo PM, West M, Montelaro RC.Monoclonal antibodies (MCAbs) were used to dissect the antigenic sites of the surface glycoproteins of the prototype cell-adapted Wyoming strain of equine infectious anemia virus (EIAV). Serologic reactivities of these MCAbs were determined by ELISA, additive ELISA, competitive ELISA, and Western blot assays. The results indicated that antigenic reactivity of gp90 was localized on at least four distinct epitopes, two of which were important in neutralization. Our studies also revealed that these epitopes were localized on overlapping antigenic sites on gp90. On the other hand, only two distinc...
Liu ZQ, Xia J, Wang GL, Kuermanali N.Hyalomma asiaticum tick, an important ectozoic parasite causes tickle, pain, anemia, weight loss, and paralysis in its hosts, which include humans, cattle, sheep, horses, camels, and hares. The 4D8 gene can be a potential vaccine candidate antigen for H. asiaticum. In the present study, we cloned and expressed the 4D8 gene of H. asiaticum from Xinjiang Province. Primers were designed according to the H. asiaticum tick 4D8 gene sequence available in GenBank. The gene was amplified by reverse transcription-polymerase chain reaction and the fragments were subcloned into the prokaryotic expression...
Hofmaier F, Hauck SM, Amann B, Degroote RL, Deeg CA.Autoimmune uveitis is a sight-threatening disease in which autoreactive T cells cross the blood-retinal barrier. Molecular mechanisms contributing to the loss of eye immune privilege in this autoimmune disease are not well understood. In this study, the authors investigated the changes in the matrix metalloproteinase network in spontaneous uveitis. Methods: Matrix metalloproteinase (MMP) MMP2, MMP9, and MMP14 expression and tissue inhibitor of metalloproteinase (TIMP)-2 and lipocalin 2 (LCN2) expression were analyzed using Western blot quantification. Enzyme activities were examined with zymog...
Koch DW, Schnabel LV, Ellis IM, Bates RE, Berglund AK.Mesenchymal stem cells (MSCs) secrete paracrine factors and extracellular matrix proteins that contribute to their ability to support tissue healing and regeneration. Both the transcriptome and the secretome of MSCs can be altered by treating the cells with cytokines, but neither have been thoroughly investigated following treatment with the specific cytokine transforming growth factor (TGF)-β2. RNA-sequencing and western blotting were used to compare gene and protein expression between untreated and TGF-β2-treated equine bone marrow-derived MSCs (BM-MSCs). A co-culture system was utilized t...
Kenney RM, Cummings MR, Teuscher C, Love CC.Testicular degeneration is a major cause of subfertility in stallions, although an aetiological diagnosis cannot be made in most cases. In the present study, autoimmune testicular degeneration was induced and evaluated in stallions by immunizing stallions with their own spermatozoa mixed with an adjuvant. The factors evaluated included changes in semen quality and testicular histology. A large decrease in sperm number and quality was observed in response to sperm autoantigens. An ELISA test specific for antisperm antibodies was developed which enabled antibody titres in serum, seminal plasma a...
Komori M, Higami A, Imai Y, Imaoka S, Funae Y.A form of P450 [termed P450(h-1)] was purified from the liver microsomes of a male horse to electrophoretic homogeneity. The specific content of the final P450(h-1) preparation was 14.8 nmol/mg of protein and the recovery was 0.38% of the microsomal P450. The apparent molecular weight of P450(h-1) was 52,000 Da. The absorption spectra of P450(h-1) indicated that P450(h-1) was a low- and high-spin mixed type P450 in the oxidized form. The reconstituted system containing P450(h-1) could catalyze benzphetamine N-demethylation, 7-ethoxycoumarin O-deethylation, and testosterone 16 alpha-hydroxylati...
Hansson E, Skiöldebrand E.To treat osteoarthritic chondrocytes and thereby reduce the inflammation with a drug combination that primarily affects 5-HT- and ATP-evoked Ca2+ signaling. In osteoarthritic chondrocytes, Ca2+ signaling is elevated, resulting in increased production of ATP and inflammatory mediators. The expression of TLR4 and Na+/K+-ATPase was used to evaluate the inflammatory status of the cells. Methods: Equine chondrocytes were collected from joints with mild structural osteoarthritic changes and cultured in monolayers. The cells were treated with a combination of bupivacaine (1 pM) and sildenafil (1 ...
Proudman CJ, Trees AJ.Whole worm extract (WWE) and excretory/secretory (E/S) antigens of Anoplocephala perfoliata were characterised by SDS-PAGE and their use in the serodiagnosis of equine cestodosis was evaluated. An enzyme-linked immunosorbent assay (ELISA) was used to compare WWE and E/S antigen as the capture layer in an antibody capture ELISA. E/S antigen gave the best differentiation between sera from tapeworm-positive and tapeworm-negative horses. The E/S-ELISA was optimised and validated against sera from horses of known tapeworm status. This assay gave a diagnostic sensitivity of 68% (n = 38) and a specif...
Snijder EJ, Wassenaar AL, Spaan WJ.To study the proteolytic processing of the equine arteritis virus (EAV) replicase open reading frame 1a (ORF1a) protein, specific antisera were raised in rabbits, with six synthetic peptides and a bacterial fusion protein as antigens. The processing of the EAV ORF1a product in infected cells was analyzed with Western blot (immunoblot) and immunoprecipitation techniques. Additional information was obtained from transient expression of ORF1a cDNA constructs. The 187-kDa ORF1a protein was found to be subject to at least five proteolytic cleavages. The processing scheme, which covers the entire OR...
Bougrine SI, Fihri OF, Fehri MM.A Western immunoblotting procedure has been developed for the detection of African horse sickness virus (AHSV) protein-specific antibody responses. This assay readily identifies antibodies specific for at least 4 distinct, AHSV proteins, including VP5, NS1, NS2 and NS3/NS3a. By using the AHSV non-structural proteins as 'markers', the Western blotting procedure could be employed to provide a reliable means of discriminating between animals vaccinated with a purified, inactivated AHSV vaccine and those either naturally infected or vaccinated with a live, attenuated AHSV vaccine.
Thougaard AV, Jaliashvili I, Christiansen M.The glycoprotein tetranectin (TN) found in human serum is a 90-kDa homotrimeric C-type lectin binding Ca2+, heparin and plasminogen kringle 4. TN is suggested as being implicated in tissue remodelling. The antigenic reactivity of putative TN was examined in serum from 14 different animal species using three sandwich enzyme immunoassays for human TN. Crab-eating macaque serum showed the strongest reaction, followed by horse and cat. Serum from cow, goat, pig, mouse and chicken reacted weakly, while dog, trout, and the amphibian and the reptile species did not react. The TN-like protein from mac...
Vidal Moreno de Vega C, Lemmens D, de Meeûs d'Argenteuil C, Boshuizen B, de Maré L, Leybaert L, Goethals K, de Oliveira JE, Hosotani G, Deforce D....Important changes in glucose transporter (GLUT) expression should be expected if the glucose influx plays a pivotal role in fuelling or connecting metabolic pathways that are upregulated in response to exercise. The aim was to assess GLUT4, 8, and 12 dynamics in response to training and acute exercise. Sixteen untrained Standardbred mares (3-4 year) performed an incremental SET at the start and end of 8 weeks harness training. M. pectoralis (PM) and M. vastus lateralis (VL) muscle biopsies were taken before and after each SET, allowing for comparing rest and acute samples in untrained (UT) ...
Lannergård J, Frykberg L, Guss B.Streptococcus equi subspecies equi is an important horse pathogenic bacterium causing a serious disease called strangles. Using bioinformatics we identified a gene denoted cne (gene encoding collagen-binding protein from S. equi) coding for a novel potential virulence factor of this species called protein CNE. The protein is composed of 657 amino acids and has the typical features found in cell surface-anchored proteins in Gram-positive bacteria. CNE displays amino acid sequence similarities to the previously well-studied collagen-binding protein CNA from Staphylococcus aureus, a proven virule...
Janda J, Plattet P, Torsteinsdottir S, Jonsdottir S, Zurbriggen A, Marti E.Allergic horses react to innocuous environmental substances by activation of Th2 cells and production of allergen-specific IgE antibodies. The mechanisms leading to Th2 differentiation are not well understood. In humans and mice, epithelial cell-derived thymic stromal lymphopoietin (TSLP) plays a central role in this process. Little is known about equine TSLP (eqTSLP) and its role in allergic diseases and our current knowledge is limited to the assessment of TSLP mRNA expression. In order to be able to study eqTSLP at the protein level, the aim of the present study was to produce recombinant e...
Mar Htun Z, Laikul A, Pathomsakulwong W, Yurayart C, Lohnoo T, Yingyong W, Kumsang Y, Payattikul P, Sae-Chew P, Rujirawat T, Jaturapaktrarak C....Pythium insidiosum causes a life-threatening infection termed pythiosis in humans and other animals. The organism has been identified in tropical and subtropical environments worldwide. Since 1985, human pythiosis has been increasingly reported from Thailand. Seroprevalence studies estimated that 32,000 Thai people had been exposed to the pathogen. In 2018, the first animal pythiosis case in Thailand was diagnosed in a horse. Here, we investigated the seroprevalence of anti-P. insidiosum antibodies in the Thai equine population. Methods: We surveyed serum anti-P. insidiosum antibodies in 15...
Daraban Bocaneti F, Altamura G, Corteggio A, Tanase OI, Dascalu MA, Pasca SA, Hritcu O, Mares M, Borzacchiello G.Bovine papillomaviruses -1/-2 (BPVs) are small non-enveloped double-stranded DNA viruses able to infect the skin of bovids and equids, causing development of neoplastic lesions such as bovine cutaneous fibropapillomas and equine sarcoid. Matrix metalloproteinases (MMPs) are a group of zinc-dependent endopeptidases that degrade basal membrane and extracellular matrix, whose function is essential in physiological processes such as tissue remodeling and wound healing. MMPs activity is finely regulated by a balancing with expression of tissue inhibitors of MMPs (TIMPs), a process that is impaired ...
Frey J, Eberle S, Stahl C, Mazuet C, Popoff M, Schatzmann E, Gerber V, Dungu B, Straub R.In Europe the incidence of botulism in horses has increased in the last decade due to the growing popularity of haylage feeding. Recombinant vaccines are safer and less expensive to produce and are generally better tolerated than toxoids. Objective: To investigate whether the recombinant C-terminal half of the heavy chain of the botulinum neurotoxin C (Hc BoNT/C) in combination with an immunstimulatory adjuvant is an appropriate vaccine candidate for horses by testing its efficacy to induce neutralising antibodies and by comparing its immunogenic properties and adverse reactions to a commercia...
Xiao C, Liu Y, Jiang Y, Magoffin DE, Guo H, Xuan H, Wang G, Wang LF, Tu C.Four monoclonal antibodies (mAbs) were generated by immunizing BALB/C mice with recombinant nucleocapsid protein (N) of Nipah virus (NiV) and Hendra virus (HeV) expressed in E. coli. Two mAbs each were obtained for the HeV N and NiV N, respectively. All four mAbs displayed specific reactivity with the recombinant N proteins of both viruses by western blot, which was further confirmed by immunofluorescent antibody assay using fixed insect cells infected with recombinant baculoviruses expressing either the HeV or NiV N protein. Epitope mapping using a 12-mer random peptide phage display library ...
Hornyák A, Dénes B, Szeredi L, Dencsö L, Rusvai M.Two monoclonal antibodies against the Bucyrus strain of equine arteritis virus (EAV) were produced, and according to immunoperoxidase reaction following Western blot of electrophoresed EAV structural proteins, they recognized the nucleocapsid (N) protein antigen (14-kDa protein). Besides reacting with the blotted polypeptide, the antibodies of the two clones (designated 1H1 and 4G6) selected from 576 have shown high affinity and specificity to intracellular virus antigen as well. Both antibodies reacted with the representatives of the different subtypes of equine arteritis virus providing a su...
van Maanen C, Vreeswijk J, Moonen P, Brinkhof J, de Boer-Luijtze E, Terpstra C.Ten monoclonal antibodies (MAbs) were produced against equine herpes virus type 1 (EHV1). Two appeared type-specific, while the other eight were directed against epitopes common to both EHV1 and EHV4. Two MAbs directed against the glycoprotein gp2 recognized linear epitopes, as demonstrated by Western blotting. With pools of type-specific MAbs, 282 field isolates were typed in an immunoperoxidase monolayer assay (IPMA). From a total of 254 fetal or neonatal isolates, 244 (96%) were typed as EHV1, whereas 14 out of 15 (93%) respiratory tract isolates were typed as EHV4. Surprisingly, 3 out of 1...
Wang LF, Gould AR, Selleck PW.Full-length cDNA clones coding for the matrix (M) and fusion (F) proteins of equine morbillivirus (EMV) were isolated by RT-PCR, and expressed in Escherichia coli using two different expression systems. Western blot analysis indicated that the M and F proteins, expressed either by itself or as fusion proteins with glutathione S-transferase (GST), were insoluble and degraded after expression. Analysis of the degradation pattern of recombinant M protein suggested that the N-terminus of the matrix protein might be more stable and antigenic than the C-terminal region. Therefore a third system was ...
Deeg CA, Hauck SM, Amann B, Kremmer E, Stangassinger M, Ueffing M.Equine recurrent uveitis (ERU) is a valuable model for autoimmune diseases, since it develops frequently and occurs spontaneously. We investigated the overall expression level of three major retinal autoantigens in normal retinas and various ERU stages. Analysis of retinal proteomes of both, healthy and diseased retinas revealed an almost unaffected expression of IRBP, S-antigen and cRALBP in ERU cases. Validation of these findings with western blots and immunohistochemistry confirmed constant to increased expression of these autoantigens, although loss of their physiological expression sites ...
Steelman SM, Chowdhary BP.Equine laminitis is a devastating disease that causes severe pain in afflicted horses and places a major economic burden on the horse industry. In acute laminitis, the disintegration of the dermal-epidermal junction can cause the third phalanx to detach from the hoof wall, leaving the horse unable to bear weight on the affected limbs. Horses that survive the acute phase transition into a chronic form of laminitis, which is often termed "founder". Some evidence suggests that chronic laminar inflammation might be associated with alterations in the endocrine and immune systems. We investigated th...
Wu D, Murakami K, Liu N, Konishi M, Muneta Y, Inumaru S, Kokuho T, Sentsui H.The equine interleukin-18 (IL-18) cDNA that contains the coding sequence was cloned and a recombinant baculovirus, named AcEIL-18, was constructed. The recombinant protein of the equine IL-18 was expressed by AcEIL-18 and its expression was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Insect cells infected with AcEIL-18 secreted a precursor IL-18 with 24 kilo dalton (kDa) into the culture supernatant. Western blot analysis showed that mature equine IL-18 about 18 kDa was also confirmed without co-expression of caspase-1. Culture supern...
