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.
Marsh AE, Barr BC, Madigan J, Lakritz J, Nordhausen R, Conrad PA.Neosporosis was diagnosed in an 11-year-old Quarter Horse gelding with clinical signs and diagnostic test results compatible with equine protozoal myeloencephalitis (EPM). Presumptive postmortem diagnosis of EPM attributable to Sarcocystis neurona infection is generally made on the basis of detecting an antibody titer to S neurona in the CSF or characteristic histologic lesions, even when parasites have not been specifically identified. Neosporosis was confirmed in the horse described here by use of immunohistochemical examination, in vitro culturing, and ultrastructural and molecular characte...
Tákai S, Hidaka D, Fujii M, Shindoh Y, Murata T, Nakanishi S, Sasaki Y, Tsubaki S, Kamada M.Humoral immune responses in 16 foals to virulence-associated 15- to 17-kDa antigens of Rhodococcus equi were studied during the first fourteen weeks of life on two horse-breeding farms with a persistent incidence of R. equi infection. Serum antibody levels specific for 15- to 17-kDa antigens were measured by enzyme-linked immunosorbent assay and Western immunoblotting. Immunoglobulin G (IgG) antibodies specific to 15- to 17-kDa antigens were detected by all the foals. R. equi was found in the feces of foals during week 1 of life, and the number of fecal R. equi rapidly increased to the highest...
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...
Todhunter PG, Kincaid SA, Todhunter RJ, Kammermann JR, Johnstone B, Baird AN, Hanson RR, Wright JM, Lin HC, Purohit RC.To use lipopolysaccharide (LPS) to create synovitis in the midcarpal joint of ponies, and to assess the morphologic, histochemical, and immunohistochemical effects of synovitis on articular cartilage of the third carpal bone. Methods: 2- to 3-year-old ponies, 6 control (group 1) and 6 treated (group 2). Methods: Synovitis was induced in 1 midcarpal joint of group-2 ponies by intra-articular injections of LPS (0.02 micrograms/kg of body weight), morphine (0.1 mg/kg), and saline solution (group 2a) and a morphine and saline solution alone in the contralateral midcarpal joint (group 2b). Articula...
Lebelt J, Hagenau K.Borna disease (BD) is a naturally occurring enzootic encephalomyelitis of horses and sheep. The aetiological agent, Borna disease virus (BDV) is an unclassified, neurotropic, negative stranded RNA virus. The study aimed at providing further information on BD of naturally infected animals. Samples obtained from 20 animals (18 horses, 1 donkey, 1 sheep) were investigated by a series of virological and molecular biological tests. The highly sensitive reverse transcription-polymerase chain reaction (RT-PCR) method was used to analyze the tissue distribution of BDV-specific RNA. BDV-specific RNA wa...
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...
Weidmeyer CE, Solter PF.The Incstar(R) SPQ II human haptoglobin (Hpt) (Incstar Corporation, Stillwater, MN) immunoturbidimetric assay was validated for the determination of serum and plasma Hpt concentrations in dogs and horses. The anti-human Hpt antiserum supplied with the assay, displayed monospecificity to both dog and horse serum Hpt by immunoelectrophoresis and Western blotting techniques. The automated immunoturbidimetric assay results correlated well with the cyanmethemoglobin binding assay (r=0.953 for canine serum and r=0.941 for equine serum), and had excellent precision at both high and low serum Hpt conc...
Granstrom DE.This article reviews recent advances in laboratory diagnosis of equine parasitic diseases. Laboratory diagnosis of most equine parasitic diseases continues to rely on standard methods. Only laboratory diagnostic tests for EPM, cryptosporidiosis, and giardiasis were included. The criteria for testing and interpretation of results for each new diagnostic method were explained. Western blot and PCR testing for EPM and immunofluorescent staining with monoclonal antibodies for cryptosporidiosis and giardiasis were reviewed.
Madigan JE, Rikihisa Y, Palmer JE, DeRock E, Mott J.The original objective was to determine seroprevalence of Ehrlichia risticii antibody among horses in California. On the basis of the unexpected results of the survey, an investigation into the accuracy and reproducibility of results of the indirect fluorescent antibody (IFA) test for E risticii was carried out. Methods: Prospective, seroprevalence study. Methods: Healthy horses (n = 655) and horses with clinical signs of equine monocytic ehrlichiosis (EME; n = 514) from various regions of California. Methods: The IFA test was performed. Results were compared with results of an ELISA and with ...
Dillon AM, Heath MF.Protein tyrosine phosphorylation (PTP) in thrombin- and platelet-activating factor (PAF)-stimulated equine platelet activation was investigated in the absence and presence of 2 protein tyrosine kinase inhibitors (PTKIs), methyl 2,5-dihydroxycinnamate (MDHC) and genistein. Washed equine platelets aggregated irreversibly in response to thrombin or PAF in an agonist concentration dependent fashion. MDHC produced an MDHC concentration and time dependent inhibitory effect on rate and extent of thrombin- and PAF-induced aggregations, whereas the effect of genistein on the same parameters was only ge...
Nakamura Y, Kishi M, Nakaya T, Asahi S, Tanaka H, Sentsui H, Ikeda K, Ikuta K.Borna disease (BD) is a progressive poliomeningoencephalomyelitis which occurs naturally in horses and sheep. Here, peripheral blood mononuclear cells (PBMC) derived from 57 healthy horses in Japan were examined by a nested reverse transcription-polymerase chain reaction to determine the prevalence of BD virus (BDV) infection. Seventeen (29.8%) of the samples were positive by this examination and the specificity of the amplified product was confirmed by hybridization with authentic oligomer probes. About 60% of the BDV RNA-positive individuals also showed seropositivity by Western blotting. Th...
Pfeifer Barbosa I, Böse R, Peymann B, Friedhoff KT.Epidemiological studies of Babesia equi and B. caballi were undertaken in a herd of 120 pastured horses in Rio de Janeiro, Brazil. The area where the horses were held was shown to be highly endemic for both Babesia spp., i.e. the prevalence of B. equi antibodies in horses aged 6 months or older ranged from 90.6% to 100% as determined by the immunofluorescence antibody (IFA) test, and the prevalence of B. caballi antibodies as determined by Western blot ranged from 59.4% to 65.5%. From the herd, 20 foals and their dams were selected to estimate the degree of tick infestation and the foals were ...
Schelp C, Böse R, Micha A, Hentrich B.High-titre equine immune sera were used to screen a lambda gt 11 expression library of Babesia equi cDNA fragments. Two cDNA clones which did not cross-hybridize to each other were studied. Both clones hybridized specifically to DNA from B. equi but not to DNA from B. caballi, B. divergens or B. ovis. Recombinant proteins were expressed as glutathione S-transferase (GST) fusion proteins with apparent molecular weights of 40 kDa and 75 kDa. Polyclonal antibodies directed against the 40 kDa and 75 kDa recombinant proteins detected native antigens of 55 kDa and 50 kDa respectively in crude lysate...
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,...
Strizki JM, Repik PM.We have re-evaluated the relationships among the polypeptides of eastern equine encephalitis (EEE) viruses using SDS-PAGE and peptide mapping of individual virion proteins. Four to five distinct polypeptide bands were detected upon SDS-PAGE analysis of viruses: the E1, E2 and C proteins normally associated with alphavirus virions, as well as an additional more rapidly-migrating E2-associated protein and a high M(r) (HMW) protein. In contrast with previous findings by others, the electrophoretic profiles of the virion proteins of EEE viruses displayed a marked correlation with serotype. The pro...
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...
Moore KH, Dunbar BS, Bousfield GR, Ward DN.Inhibin has been characterized from a number of mammals; however, it has not been extensively studied in horses. Western blot analysis was used to examine the size heterogeneity of equine inhibin alpha- and beta-subunits. The distribution of equine inhibin activity from the initial sizing column (S-200, 25 x 94 cm) indicated that the majority of equine inhibin activity was present as larger-molecular-size forms. When the large forms were analyzed by Western blot in nonreducing conditions, alpha-subunit bands were detected at 40,000 M(r), 56,000 M(r), 80,000 M(r), and 90,000 M(r); beta a reacti...
Rosol TJ, Nagode LA, Robertson JT, Leeth BD, Steinmeyer CL, Allen CM.Humoral hypercalcemia of malignancy was evident in a horse that had a locally invasive ameloblastoma of the left hemimandible. Surgical removal of the neoplasm resulted in prompt return of serum calcium and parathyroid hormone concentrations to within reference limits. The tumor contained parathyroid hormone-related protein, as demonstrated by immunohistochemistry and western blot analysis. It is likely that production of this protein by the neoplasm was important in the pathogenesis of the hypercalcemia. The case represented a sporadic form of humoral hypercalcemia of malignancy attributable ...
Carter SD, May C, Barnes A, Bennett D.Antibody levels (IgG and IgM) to Borrelia burgdorferi were measured in the sera and synovial fluids of UK horses. Western blotting against B. burgdorferi was also used on samples from seropositive horses. A low incidence of seropositivity was shown in horses from most parts of the UK. This increased in areas that have a high incidence of human and canine borreliosis (Norfolk and south coast). Leptospira infections of horses did not cause cross reactions in the B. burgdorferi ELISA. Most horses did not display clinical signs of Lyme disease. As with dogs and man, it is apparent that B. burgdorf...
Böse R, Peymann B.From Babesia caballi in vitro cultures a preparation of 100% infected erythrocytes was obtained. From this, B. caballi antigens were extracted with the detergent 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) and used as ELISA antigens. A control antigen of normal erythrocytes from the same donor horse was prepared in an identical manner. The ELISA and Western blot were validated by testing of sera from horses experimentally infected with B. caballi or B. equi or not infected with Babesia spp. ELISA and Western blot results were compared with those obtained by the immunofl...
Böse R, Peymann B, Barbosa IP.Sera from 60 horses held in breeding herd in Brazil were examined monthly by ELISA, immunofluorescence antibody test (IFAT) and Western blot. All foals had maternal antibodies detectable by ELISA and IFAT, and sero-conversion took place between the 2nd and 5th month of age. The 48 and 50 kDa antigens were recognized first in the course of infection. Of 79 sera taken after sero-conversion 78 reacted with the 48 kDa antigen, 76 with the 50 kDa, 50 with the 70 kDa, 54 with the 112 kDa, 72 with the 141 kDa antigen. In general, sera from horses older than 1 year reacted with all 5 diagnostic antige...
Teuscher C, Kenney RM, Cummings MR, Catten M.In this study, 2 stallions were immunised with their own spermatozoa to ascertain whether an antisperm autoantibody response could be mounted. The results demonstrated that the stallion can recognise and respond to sperm autoantigens by producing circulating antisperm antibodies, primarily of the IgG class. Such autoantibodies appeared 2-4 weeks after inoculation and persisted for 6-20 weeks. Immunochemical characterisation by western blot identified two major sperm autoantigens, with molecular weights of 70 kD and 62 kD. Control pony stallions immunised with adjuvants alone failed to exhibit ...
Pilling A, Davison AJ, Telford EA, Meredith DM.Glycoprotein 45 is a major envelope glycoprotein of equine herpesvirus type 1. The gene encoding this protein is located between map units 0.615 and 0.636 on the virus genome and evidence has suggested that it is encoded by gene 52, one of four genes within this region. Using PCR we have amplified gene 52 and subsequently cloned it into a mammalian expression vector under the control of the human cytomegalovirus immediate early gene promoter. The gene was expressed in COS-7 cells and its product was detected by immunofluorescence and Western blotting. The results indicate that glycoprotein 45 ...
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...
Bowling AT, Penedo MC, Gordon L, Bell K.A modified procedure for detection of the two alleles of equine plasminogen using Western blotting methods following polyacrylamide gel isoelectric focusing is described. Gene frequencies in 23 breeds and Equus przewalskii are provided.
McDowell KJ, Adams MH, Williams NM.Proteins synthesized and released in vitro by oviducts collected from horse mares during estrus and at day 4 after ovulation for bred and nonbred mares were examined by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS PAGE) and fluorography. Ampullary and isthmic regions both produced a wide array of nondialyzable proteins in culture. Major proteins or groups of proteins identified according to relative molecular weight (kDa) and apparent isoelectric point (pI) were at 100 kDa, pI 8; 100-200 kDa, pI 6; 150 kDa, pI 4.5; 60-100 kDa, pI 4; and an array of polypep...
Fikrig E, Magnarelli LA, Chen M, Anderson JF, Flavell RA.Enzyme-linked immunosorbent assays (ELISA) and immunoblots using either whole-cell lysates of Borrelia burgdorferi or an antigenic region of flagellin (41-G) as the antigen were performed, and the abilities of the two assays to detect antibodies to this spirochete in dog, cottontail rabbit, and horse sera were compared. Assays using whole-cell B. burgdorferi lysates as the antigen were more sensitive for detecting antibodies. ELISA with 41-G as the antigen were specific for Borrelia antibodies but were not as sensitive as the assays with whole-cell lysates coated to the solid phase. Use of rec...
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...
Harty RN, Caughman GB, Holden VR, O'Callaghan DJ.Equine herpesvirus 1 (EHV-1, Kentucky A strain) preparations enriched for defective interfering particles (DIPs) can readily establish persistent infection. The UL1 gene, which is conserved in the genome of DIPs that mediate persistent infection, maps between nucleotides 1418 and 2192 (258 amino acids) from the L (long) terminus. UL1 has no homology with any known gene encoded by herpes simplex virus type 1 but has limited homology to open reading frame 2 of varicella-zoster virus and the "circ" gene of bovine herpesvirus type 1. Previous work showed that the EHV-1 UL1 gene belongs to the earl...
Sellon DC, Cullen JM, Whetter LE, Gebhard DH, Coggins L, Fuller FJ.An IgG1 mouse monoclonal antibody, designated 1.646, is described which recognizes a cytoplasmic antigen of equine mononuclear phagocytes. Indirect fluorescent antibody staining of peripheral blood leukocytes reveals a granular cytoplasmic staining, predominantly in adherent blood mononuclear cells. Indirect fluorescent antibody staining is positive for alveolar and peritoneal macrophages. In some horses, a few neutrophils are also stained. In equine tissue samples stained by immunohistochemistry, the distribution of positive cells is consistent with the distribution of tissue macrophages. The...
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...
Basso RM, Andrade DGA, Alves CEF, Laufer-Amorim R, Borges AS, Oliveira-Filho JP.Dwarfism is a skeletal disorder that causes abnormal growth. In Miniature horses, dwarfism can occur as chondrodysplastic dwarfism, an autosomal recessive disorder associated with five mutations (D1, D2, D3*, D4 and c.6465A > T variant) in the aggrecan (ACAN) gene. The aim of this study was to evaluate the expression of aggrecan (at the gene and protein level) and specific cytokines (IL-1β, IL-6, and TNF-α) in the articular cartilage of Miniature horses with chondrodysplastic dwarfism (D4/c.6465A > T genotype). Metatarsal bone samples from eight dwarf Miniature horses were collected fo...
Nagy AM, Sathe SR, Atta AH, Hammam AMM, Hsu WH.In equine parturition, the role of progestins along with the nuclear progesterone receptor (nPR) signaling pathway in the placenta is not completely clarified. The progestins play an integral role in maintaining myometrial quiescence during the late stage of pregnancy via acting on nPR isoforms (PRA and PRB; PRB is more active than PRA). The current study aimed to determine the PRA and PRB expressions in the term equine placenta at the gene and protein levels. Six term equine placentas were used in this study. Reverse transcription polymerase chain reaction (RT-PCR) was used to quantify the mR...
Watson ED, Thomson SR, Howie AF.A steroidogenic acute regulatory (StAR) protein has been identified in several species as a probable important rate-limiting step in steroidogenesis. This protein is believed to be responsible for transporting cholesterol from the outer to the inner mitochondrial membrane. It is known that equine chorionic gonadotrophin (eCG) stimulates steroidogenesis in the corpora lutea of early pregnant mares and that eCG also upregulates StAR mRNA in bovine ovaries. In the present study, ovarian tissue from cyclic and early pregnant mares was immunostained to detect the distribution of the StAR protein. W...
Qi T, Hu Y, Hu Z, Zhao S, Cullinane A, Lyons P, Gildea S, Wang X.Quantitation of virions is one of the important indexes in virological studies. To establish a sensitive and rapid quantitative detection method for equine arteritis virus (EAV), an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed by using two EAV nucleoprotein monoclonal antibodies (mAbs), 2B9 and 2B3, prepared in this study. After condition optimization, mAb 2B9 was used as the capture antibody, and HRP-labeled 2B3 was chosen as the detecting antibody. The AC-ELISA had a good standard curve when viral particles of the Bucyrus EAV strain were used as a reference stan...
Schelp C, Böse R, Micha A, Hentrich B.High-titre equine immune sera were used to screen a lambda gt 11 expression library of Babesia equi cDNA fragments. Two cDNA clones which did not cross-hybridize to each other were studied. Both clones hybridized specifically to DNA from B. equi but not to DNA from B. caballi, B. divergens or B. ovis. Recombinant proteins were expressed as glutathione S-transferase (GST) fusion proteins with apparent molecular weights of 40 kDa and 75 kDa. Polyclonal antibodies directed against the 40 kDa and 75 kDa recombinant proteins detected native antigens of 55 kDa and 50 kDa respectively in crude lysate...
Lambertini C, Zannoni A, Romagnoli N, Bombardi C, Morini M, Dondi F, Bernardini C, Forni M, Rinnovati R, Spadari A.Large colon volvulus in horses is associated with a poor prognosis, especially when ischemic-reperfusion injury of the affected intestinal tract develops. Proteinase-activated receptor 2 (PAR2) plays an important role in the pathogenesis of inflammation in the gastrointestinal tract. The aim of this study was to evaluate the distribution and expression of PAR2 in colonic pelvic flexure of horses spontaneously affected by large colon volvulus (CVH group). Eight horses admitted for severe abdominal colon volvolus and which underwent surgery were included. Colon samples were collected after enter...
Milillo P, Traversa D, Elia G, Otranto D.Larvae of Rhinoestrus spp. (Diptera, Oestridae) infect nasal and sinus cavities of horses, causing a nasal myiasis characterized by severe respiratory distress. Presently, the diagnosis of horse nasal botfly relies on the observation of clinical signs, on the post mortem retrieval of larvae or on molecular assays performed using pharyngeal swabs. The present study was carried out to characterize larval somatic proteins and salivary glands of Rhinoestrus spp. in a preliminary assessment towards the immunodiagnosis of equine rhinoestrosis. Out of the 212 necropsied horses 13 were positive for th...
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...
de Arruda Coutinho LC, de Jesus AL, de Paiva Fontes KF, Coimbra EC, Mariz FC, de Freitas AC, de Cássia Carvalho Maia R, de Castro RS.Equine Infectious Anemia (EIA) is a persistent lentivirus infection of horses which causes a chronic clinical condition with worldwide importance in veterinary medicine. The p26 protein is usually prepared for use as an antigen in serological tests for EIA diagnosis since it is a well-conserved gene sequence and very immunogenic. In view of the ability of yeast to make post-translational modifications of proteins, this study was carried out to allow Pichia pastoris to be used for the expression of a synthetic codon-optimized EIAV p26 gene. The gene was cloned into pPICZαA vector after appropr...
Nebbia C, Dacasto M, Carletti M.Little is known about the effects of aging on the hepatic drug metabolizing capacity of horses despite the relatively long lifespan characterizing this species. A wide array of cytochrome P450 (CYP)-dependent monooxygenases, carboxylesterases and transferases were assayed in liver microsomes from 50 female horses in an age range between less than 1 year to over 12 years. Rather unexpectedly, both the CYP content and the activity of NADPH cytochrome c reductase rose as a function of age. Accordingly, a general increasing trend was recorded in the rate of the in vitro metabolism of the substrate...
Rossano MG, Schott HC, Kaneene JB, Murphy AJ, Kruttlin EA, Hines MT, Sellon DC, Patterson JS, Elsheikha HM, Dubey JP, Mansfield LS.To determine whether daily administration of pyrantel tartrate can prevent infection in horses experimentally challenged with Sarcocystis neurona. Methods: 24 mixed-breed specific-pathogen-free weanling horses, 10 adult horses, 1 opossum, and 6 mice. Methods: Sarcocystis neurona-naïve weanling horses were randomly allocated to 2 groups. Group A received pyrantel tartrate at the labeled dose, and group B received a nonmedicated pellet. Both groups were orally inoculated with 100 sporocysts/d for 28 days, 500 sporocysts/d for 28 days, and 1000 sporocysts/d for 56 days. Blood samples were collec...
Yamaguchi K, Sawada T, Yamane S, Haga S, Ikeda K, Igata-Yi R, Yoshiki K, Matsuoka M, Okabe H, Horii Y, Nawa Y, Waltrip RW, Carbone KM.Borna disease virus (BDV) is a neurotropic pathogen that infects a wide variety of vertebrates. We have developed a new electrochemiluminescence immunoassay (ECLIA) for the detection of antibodies to BDV, using three synthetic peptides corresponding to the amino acid residues 3-20 and 338-358 of p40 and 59-79 of p24 peptide of BDV. Using the ECLIA, we examined serum samples for the presence of anti-BDV antibodies in 20 rats (experimentally BDV-infected and uninfected) and 38 horses (13 US horses, experimentally infected and uninfected, and 25 Japanese horses, feral and domestic). The ECLIA, pe...
Fernandes I, Lima EX, Takehara HA, Moura-da-Silva AM, Tanjoni I, Gutiérrez JM.Horse IgG isotypes and cross-neutralization of two snake antivenoms produced in Brazil and Costa Rica. Toxicon 000-000. This work compared the specificity, ELISA titers and IgG subclass content of the polyvalent antivenom (anti-Bothrops asper, Crotalus durissus durissus and Lachesis muta stenophrys) of Instituto Clodomiro Picado (Costa Rica) and the bothropic antivenom (anti-Bothrops jararaca, B. jararacussu, B. moojeni, B. neuwiedi and B. alternatus) of Instituto Butantan (Brazil). The role of IgG(T) and IgGa subclasses in neutralization of some venom toxic activities and the cross neutraliza...
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...
Hu WG, Alvi AZ, Fulton RE, Suresh MR, Nagata LP.A recombinant gene encoding a single-chain variable fragment (scFv) antibody against Venezuelan equine encephalitis virus (VEE) was cloned into a prokaryotic T7 RNA polymerase-regulated expression vector. A streptavidin-binding peptide gene fused to a 6His tag was attached downstream to the scFv gene. The recombinant fusion protein was expressed in bacteria as inclusion bodies that were subsequently solubilized with 8 M urea and renatured by an arginine system. Purification of the fusion protein was achieved by immobilized metal affinity chromatography. Enzyme-linked immunosorbent assay (ELISA...
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....
Strazzullo M, Corteggio A, Altamura G, Francioso R, Roperto F, D'Esposito M, Borzacchiello G.Sarcoids are peculiar equine benign tumours. Their onset is associated with Bovine Papillomavirus type -1 or -2 (BPV-1/2) infection. Little is known about the molecular interplay between viral infection and neoplastic transformation. The data regarding papillomavirus infections in human species show the inactivation of a number of tumour suppressor genes as basic mechanism of transformation. In this study the putative role of the tumour suppressor gene Fragile Histidine Triad (FHIT) in sarcoid tumour was investigated in different experimental models. The expression of the oncosuppressor protei...
Cook AG, Maxwell VB, Donaldson LL, Parker NA, Ward DL, Morrow JK.To determine whether antibodies against Sarcocystis neurona could be detected in CSF from clinically normal neonatal (2 to 7 days old) and young (2 to 3 months old) foals. Methods: Prospective study. Methods: 15 clinically normal neonatal Thoroughbred foals. Methods: Serum and CSF samples were obtained from foals at 2 to 7 days of age and tested for antibodies against S. neurona by means of western blotting. Serum samples from the mares were also tested for antibodies against S. neurona. Additional CSF and blood samples were obtained from 5 foals between 13 and 41 days after birth and between ...
Sellon DC, Cullen JM, Whetter LE, Gebhard DH, Coggins L, Fuller FJ.An IgG1 mouse monoclonal antibody, designated 1.646, is described which recognizes a cytoplasmic antigen of equine mononuclear phagocytes. Indirect fluorescent antibody staining of peripheral blood leukocytes reveals a granular cytoplasmic staining, predominantly in adherent blood mononuclear cells. Indirect fluorescent antibody staining is positive for alveolar and peritoneal macrophages. In some horses, a few neutrophils are also stained. In equine tissue samples stained by immunohistochemistry, the distribution of positive cells is consistent with the distribution of tissue macrophages. The...
Eizema K, van der Wal DE, van den Burg MM, de Jonge HW, Everts ME.During early postnatal development, the myosin heavy chain (MyHC) expression pattern in equine gluteus medius muscle shows adaptation to movement and load,resulting in a decrease in the number of fast MyHC fibers and an increase in the number of slow MyHC fibers. In the present study we correlated the expression of MyHC isoforms to the expression of sarcoplasmic(endo)reticulum Ca2+-ATPase 1 and 2a (SERCA), phospholamban (PLB), calcineurin A (CnA), and calcineurin B (CnB). Gluteus medius muscle biopsies were taken at 0, 2, 4, and 48 weeks and analyzed using immunofluorescence. Both SERCA isofor...
Porter RM, Akers RM, Howard RD, Forsten-Williams K.Interleukin-1 (IL-1) and insulin-like growth factor-I (IGF-I), which have opposing effects on matrix metabolism within articular cartilage, are thought to play prominent roles in the pathogenesis of osteoarthritis. To better understand the link between these anabolic (IGF-I) and catabolic (IL-1) stimuli, we examined exogenous IL-1 regulation of the IGF-I signaling system of articular chondrocytes (ACs). Equine ACs from non-arthritic stifle joints were expanded in monolayer culture, encapsulated for 10 days in alginate beads, and stimulated as high-density monolayers with recombinant equine IL-...
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 ...
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) ...
Mykkänen AK, Pösö AR, McGowan CM, McKane SA.In exercising horses, up to 50% of blood lactate is taken up into red blood cells (RBCs). Lactate transporter proteins MCT1, MCT2 and CD147 (an ancillary protein for MCT1) are expressed in the equine RBC membrane. In Standardbreds (SB), lactate transport activity is bimodally distributed and correlates with the amount of MCT1 and CD147. About 75% of SB studied have high lactate transport activity in RBCs. In other breeds, the distribution of lactate transport activity is unknown. Objective: To study whether similar bimodal distribution of MCT1 and CD147 is present also in the racing Finnhorse ...
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...
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...
Xu J, Cai J, Peek SF, Suresh M, Darien BJ.P-selectin glycoprotein ligand (PSGL-1) is a widely distributed adhesion molecule that plays a critical role in regulating lymphocyte homing and leukocyte trafficking during inflammation. The lack of specific reagents for equine PSGL-1 (ePSGL-1) has prevented mechanistic studies regarding its function and regulation in the horse. We synthesized a ePSGL-1 peptide to generate a monoclonal antibody (mAb), ePL1. Using flow cytometry and Western blot, we showed that ePL1 binds specifically to ePSGL-1 in transfected mammalian cells. We also demonstrated that ePL1 binds to equine leukocytes and recog...
