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.
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...
Misumi K, Tagami M, Kamimura T, Miyakoshi D, Helal IE, Arai K, Fujiki M.To quantify the urinary concentration of cartilage oligomeric matrix protein (COMP), and to evaluate the relationship between urinary COMP concentration and the catabolic activity of synovial fluid (SF) in diseased horses. Methods: COMP in horse urine was detected by immunoblotting with a monoclonal antibody (mAb; 14G4) raised against equine COMP from articular cartilage. Urine and serum samples were obtained from 83 Thoroughbred horses with aseptic joint diseases (AJD, 79 horses) or septic joint diseases (SJD, four horses) at the time of anesthesia induction, and samples of SF were obtained d...
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...
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...
Makrai L, Kira K, Kono A, Sasaki Y, Kakuda T, Tsubaki S, Fodor L, Varga J, Taka S.The plasmid profiles of virulent Rhodococcus equi strains isolated on three horse-breeding farms located in different parts of Hungary were investigated. From 49 soil samples collected on the three farms, 490 R. equi isolates (10 from each sample) were obtained and tested for the presence of 15- to 17-kDa antigens (VapA) by immunoblotting and PCR. Ninety-eight VapA-positive isolates were detected from 30 of the 49 culture-positive samples with a prevalence ranging from 13.1% to 23.2%. Of the 98 virulent isolates, 70 contained an 85-kb type I plasmid, 13 contained an 87-kb type I plasmid, and 1...
Simonen-Jokinen T, Maisi P, Tervahartiala T, McGorum B, Pirie S, Sorsa T.Matrix metalloproteinases (MMPs) are involved in tissue destruction in allergic airway diseases. We studied the ability of various allergenic substances to directly activate recombinant 92kDa proMMP-9. The substances included hay dust suspension (HDS) and its components (supernatant, particulate matter and wash fluid of particulate matter), storage mite extract and two Aspergillus fumigatus extracts. The allergen suspensions were incubated in vitro with proMMP-9. After incubation the conversion of proMMP-9 to 10kDa lower active forms were studied using gelatin zymography and Western immunoblot...
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...
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...
Kankavi O, Roberts MS.Once considered unique to the lung, surfactant proteins have been clearly identified in the intestine and peritoneum and are suggested to exist in several other organs. In the lung, surfactant proteins assist in the formation of a monolayer of surface-active phospholipid at the liquid-air interface of the alveolar lining, reducing the surface tension at this surface. In contrast, surface-active phospholipid adsorbed to articular surfaces has been identified as the load-bearing boundary lubricant of the joint. This raises the question of whether surfactant proteins in synovial fluid (SF) are re...
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...
Lillich JD, Rakestraw PC, Roussel AJ, Finley MR, Ganta S, Freeman LC.To determine whether ether-a-go-go (ERG) potassium channels are expressed in equine gastrointestinal smooth muscle, whether ERG channel antagonists affect jejunal muscle contraction in vitro, and whether plasma cisapride concentrations in horses administered treatment for postoperative ileus (POI) are consistent with ERG channels as drug targets. Methods: Samples of intestinal smooth muscle obtained from 8 horses free of gastrointestinal tract disease and plasma samples obtained from 3 horses administered cisapride for treatment of POI. Methods: Membranes were prepared from the seromuscular la...
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 ...
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...
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...
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...
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...
Takai S, Henton MM, Picard JA, Guthrie AJ, Fukushi H, Sugimoto C.The prevalence of virulent Rhodococcus equi in soil isolates from two horse farms in South Africa and nine clinical isolates from six foals, a foal foetus, a dog, and a monkey was investigated. The isolates were tested for the presence of virulence plasmid DNA and 15- to 17-kDa antigens by immunoblotting. Rhodococcus equi was isolated from almost all of the soil samples obtained from the two farms with 5.0 x 10(1) to 3.3 x 10(4) colony forming units per gram of soil. Virulent R. equi was isolated from three soil samples from one of the farms and appeared in 3.8% (three of 80 isolates), but not...
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.
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...
Marsh AE, Johnson PJ, Ramos-Vara J, Johnson GC.Little information is available about antigenic variation of Sarcocystis neurona isolated from horses with equine protozoal myeloencephalitis, nor is there much information available on the specific antibody pattern to S. neurona antigens of horses from different geographic regions where S. neurona isolates have been obtained. This communication reports on the characterization of a new S. neurona isolate, SN-MU1. The isolate was obtained from a 3-year old Thoroughbred that had asymmetrical neurological signs and localized skeletal muscle atrophy. This S. neurona isolate is similar to other S. ...
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...
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 ...
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 ...
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...
Minelli A, Allegrucci C, Mezzasoma I, Ronquist G, Lluis C, Franco R.Membrane vesicles of horse seminal plasma present at their surface a highly specific serine-type protease, dipeptidyl peptidase IV/CD26, a surface antigen known to characterize human prostasomes. Horse sperm cells expressed at their surface A(1) adenosine receptors (A(1)AR) and ecto-adenosine deaminase (ecto-ADA), both detected by immunoblot analysis, whereas CD26 was visualized at the equatorial segment by immunofluorescence microscopy. In addition to CD26, horse membrane vesicles showed ecto-ADA. The fusion process between horse sperm cells and vesicles was evidenced by confocal microscopy, ...
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...
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...
Hullinger PJ, Wilson WD, Rossitto PV, Patton JF, Thurmond MC, MacLachlan NJ.To determine rate of decay of passively acquired antibodies in Standardbred foals on a farm with a high seroprevalence to equine arteritis virus (EAV) and to determine whether vertical or horizontal transmission of the virus was responsible for infection on the farm. Methods: Repeated-measures study. Methods: 46 Standardbred horses (15 brood mares and their foals, 5 stallions, and 11 young horses). Methods: Serum samples obtained from horses on the farm were evaluated by serum neutralization and western immunoblot analysis to detect EAV-specific antibodies. The half-life of passively acquired ...
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...
Marsh AE, Johnson PJ, Ramos-Vara J, Johnson GC.Little information is available about antigenic variation of Sarcocystis neurona isolated from horses with equine protozoal myeloencephalitis, nor is there much information available on the specific antibody pattern to S. neurona antigens of horses from different geographic regions where S. neurona isolates have been obtained. This communication reports on the characterization of a new S. neurona isolate, SN-MU1. The isolate was obtained from a 3-year old Thoroughbred that had asymmetrical neurological signs and localized skeletal muscle atrophy. This S. neurona isolate is similar to other S. ...
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...
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 ...
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...
Takai S, Henton MM, Picard JA, Guthrie AJ, Fukushi H, Sugimoto C.The prevalence of virulent Rhodococcus equi in soil isolates from two horse farms in South Africa and nine clinical isolates from six foals, a foal foetus, a dog, and a monkey was investigated. The isolates were tested for the presence of virulence plasmid DNA and 15- to 17-kDa antigens by immunoblotting. Rhodococcus equi was isolated from almost all of the soil samples obtained from the two farms with 5.0 x 10(1) to 3.3 x 10(4) colony forming units per gram of soil. Virulent R. equi was isolated from three soil samples from one of the farms and appeared in 3.8% (three of 80 isolates), but not...
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...
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 ...
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....
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...
Minelli A, Allegrucci C, Mezzasoma I, Ronquist G, Lluis C, Franco R.Membrane vesicles of horse seminal plasma present at their surface a highly specific serine-type protease, dipeptidyl peptidase IV/CD26, a surface antigen known to characterize human prostasomes. Horse sperm cells expressed at their surface A(1) adenosine receptors (A(1)AR) and ecto-adenosine deaminase (ecto-ADA), both detected by immunoblot analysis, whereas CD26 was visualized at the equatorial segment by immunofluorescence microscopy. In addition to CD26, horse membrane vesicles showed ecto-ADA. The fusion process between horse sperm cells and vesicles was evidenced by confocal microscopy, ...
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...
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...
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...
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...
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...
Lillich JD, Rakestraw PC, Roussel AJ, Finley MR, Ganta S, Freeman LC.To determine whether ether-a-go-go (ERG) potassium channels are expressed in equine gastrointestinal smooth muscle, whether ERG channel antagonists affect jejunal muscle contraction in vitro, and whether plasma cisapride concentrations in horses administered treatment for postoperative ileus (POI) are consistent with ERG channels as drug targets. Methods: Samples of intestinal smooth muscle obtained from 8 horses free of gastrointestinal tract disease and plasma samples obtained from 3 horses administered cisapride for treatment of POI. Methods: Membranes were prepared from the seromuscular la...
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...
Misumi K, Tagami M, Kamimura T, Miyakoshi D, Helal IE, Arai K, Fujiki M.To quantify the urinary concentration of cartilage oligomeric matrix protein (COMP), and to evaluate the relationship between urinary COMP concentration and the catabolic activity of synovial fluid (SF) in diseased horses. Methods: COMP in horse urine was detected by immunoblotting with a monoclonal antibody (mAb; 14G4) raised against equine COMP from articular cartilage. Urine and serum samples were obtained from 83 Thoroughbred horses with aseptic joint diseases (AJD, 79 horses) or septic joint diseases (SJD, four horses) at the time of anesthesia induction, and samples of SF were obtained d...
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...
Kankavi O, Roberts MS.Once considered unique to the lung, surfactant proteins have been clearly identified in the intestine and peritoneum and are suggested to exist in several other organs. In the lung, surfactant proteins assist in the formation of a monolayer of surface-active phospholipid at the liquid-air interface of the alveolar lining, reducing the surface tension at this surface. In contrast, surface-active phospholipid adsorbed to articular surfaces has been identified as the load-bearing boundary lubricant of the joint. This raises the question of whether surfactant proteins in synovial fluid (SF) are re...
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...
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 ...
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...
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...
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.
Hullinger PJ, Wilson WD, Rossitto PV, Patton JF, Thurmond MC, MacLachlan NJ.To determine rate of decay of passively acquired antibodies in Standardbred foals on a farm with a high seroprevalence to equine arteritis virus (EAV) and to determine whether vertical or horizontal transmission of the virus was responsible for infection on the farm. Methods: Repeated-measures study. Methods: 46 Standardbred horses (15 brood mares and their foals, 5 stallions, and 11 young horses). Methods: Serum samples obtained from horses on the farm were evaluated by serum neutralization and western immunoblot analysis to detect EAV-specific antibodies. The half-life of passively acquired ...
Simonen-Jokinen T, Maisi P, Tervahartiala T, McGorum B, Pirie S, Sorsa T.Matrix metalloproteinases (MMPs) are involved in tissue destruction in allergic airway diseases. We studied the ability of various allergenic substances to directly activate recombinant 92kDa proMMP-9. The substances included hay dust suspension (HDS) and its components (supernatant, particulate matter and wash fluid of particulate matter), storage mite extract and two Aspergillus fumigatus extracts. The allergen suspensions were incubated in vitro with proMMP-9. After incubation the conversion of proMMP-9 to 10kDa lower active forms were studied using gelatin zymography and Western immunoblot...
Makrai L, Kira K, Kono A, Sasaki Y, Kakuda T, Tsubaki S, Fodor L, Varga J, Taka S.The plasmid profiles of virulent Rhodococcus equi strains isolated on three horse-breeding farms located in different parts of Hungary were investigated. From 49 soil samples collected on the three farms, 490 R. equi isolates (10 from each sample) were obtained and tested for the presence of 15- to 17-kDa antigens (VapA) by immunoblotting and PCR. Ninety-eight VapA-positive isolates were detected from 30 of the 49 culture-positive samples with a prevalence ranging from 13.1% to 23.2%. Of the 98 virulent isolates, 70 contained an 85-kb type I plasmid, 13 contained an 87-kb type I plasmid, and 1...
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...
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...
