Laboratory methods in equine research encompass a variety of techniques and procedures used to analyze biological samples from horses to study health, disease, genetics, and physiology. These methods include hematological analyses, biochemical assays, molecular biology techniques, and microbiological cultures. Commonly utilized laboratory techniques involve blood tests for complete blood count (CBC) and serum chemistry, polymerase chain reaction (PCR) for genetic and infectious disease studies, and enzyme-linked immunosorbent assays (ELISA) for detecting specific proteins or antibodies. These methods provide valuable data that contribute to understanding equine health and disease mechanisms. This page compiles peer-reviewed research studies and scholarly articles that explore the application, development, and outcomes of laboratory methods in the context of equine research.
Hara S, Diesterbeck US, König S, Czerny CP.The present study analyzed equine λ-light chain genes (IGLV and IGLC) transcribed in the horse breeds Rhenish-German Coldblood (RGC) and Hanoverian Warmblood (HW). Primers were generated for the major expressed IGLV subgroup 8. The significant majority of the sequences represented IGLC6/7. In RGC, IGLC1 and IGLC5 were observed in significant higher frequencies than IGLC4. In HW, significant differences were obtained for the transcription of IGLC1 and IGLC5. IGLC4 was not determined in this breed. Five allotypic IGLC1 variants, four allotypic IGLC5 variants, and three allelic as well as two al...
Vidyashankar AN, Hanlon BM, Kaplan RM.Anthelmintic resistance (AR) is a serious problem for the control of equine gastrointestinal nematodes, particularly in the cyathostomins. The fecal egg count reduction test (FECRT) is the most common method for diagnosing AR and serves as the practical gold standard. However, accurate quantification of resistance and especially accurate diagnosis of emerging resistance to avermectin/milbemycin (A/M) drugs, is hampered by a lack of accepted standards for study design, data analysis, and data interpretation. In order to develop rational evidence-based standards for diagnosis of resistance, one ...
Matthews JB, McArthur C, Robinson A, Jackson F.Cyathostomins are the primary parasitic pathogens of equids. For over 40 years, these nematodes have been controlled using broad spectrum anthelmintics. Three classes of anthelmintic are currently available for this use but, unfortunately, resistance to each of these has now been recorded in cyathostomin populations. As part of an optimal strategy to control cyathostomin infections in the field, it will be important to identify drug-resistant worms at as early a stage as possible. This objective needs to be supported by methodologies that will allow the accurate comparison of anthelmintic resi...
Papa FO, Felício GB, Melo-Oña CM, Alvarenga MA, De Vita B, Trinque C, Puoli-Filho JN, Dell'Aqua JA.The objective of this study was to determine whether replacing the egg yolk with soybean lecithin in the Botu-Crio® cryodiluent would maintain the fertility of cryopreserved stallion sperm. Two experiments were performed to evaluate cell freezability. In experiment 1, sperm from 15 stallions were frozen in Botu-Crio® (BC) or Botu-Crio® which contained 45g/L soybean lecithin (BCLS45) in place of the egg yolk. In experiment 2, we compared different concentrations of soybean lecithin: 0, 10.0, 12.5, 15.0, 17.5 and 20.0g/L (BC, BCLS10, BCLS12.5, BCLS17.5 and BCLS20, respectively). In experiment...
Lara PC, Fabrino HJ, Germano A, Silva JB.A method for the determination of As, Cd and Pb in bovine, equine and poultry liver by ICP-MS was developed and validated. Samples were digested in a microwave oven using a 10% HNO(3) solution. A set of experiments was made according to a central composite design (CCD) for optimisation of the plasma argon flow, nebuliser argon flow and radiofrequency power applied to the plasma. During the validation, Rh and Ru were evaluated as internal standards and, after validation, the best was Rh for Pb and Cd analysis, but for As better results were obtained without an internal standard. The method allo...
Griffeth GC, Callori N, Rankin SC, Boston RC, Morris DO.Meticillin-resistant Staphylococcus aureus (MRSA) causes serious skin and soft-tissue infections of humans and animals. Multiple strains of MRSA have been characterized, and one in particular, designated as strain USA 500, causes infections predominantly of horses and the people who work with them. The purpose of this study was to optimize an assay which could subsequently be used to compare the relative avidity of different S. aureus strains for equine corneocytes. Corneocytes were collected from the perineal skin of 10 healthy horses onto adhesive discs. The discs were then incubated at 37°...
de Leon PM, Campos VF, Dellagostin OA, Deschamps JC, Seixas FK, Collares T.In this study, polymerase chain reaction (PCR) reamplification of the first PCR product (2nd-PCR) and a qPCR assay were used to detect the sex determining region Y (SRY) gene from circulating cell-free fetal DNA (ccffDNA) in blood plasma of pregnant mares to determine fetal sex. The ccffDNA was isolated from plasma of 20 Thoroughbred mares (5-13 y old) in the final 3 mo of pregnancy (fetal sex was verified after foaling). For controls, plasma from two non-pregnant mares and two virgin mares were used, in addition to the non-template control. The 182 bp nucleotide sequence corresponding to the ...
Violini S, Gorni C, Pisani LF, Ramelli P, Caniatti M, Mariani P.Stem cells represent an important tool in veterinary therapeutic field such as tissue engineering. In the present study, equine amnion-derived mesenchymal stromal cells were investigated for applications in veterinary science as an alternative source to bone marrow mesenchymal stem cells and adipose stem cells. Amnion stromal cells isolation and characterization protocol is described; the in vitro cell growth rate was calculated by measuring viable cell number over 20 days. The expression of stem cell markers such as Oct-4, Nanog, Sox-2 and CD105 was assessed by retrotranscription quantitativ...
Garcia P, Paris AC, Leufroy A, Popot MA, Bonnaire Y.A quantitative method, using LC/ESI-MS(n) with a quadrupole linear ion trap mass analyzer, has been developed for the analysis of ipratropium cation in horse plasma and urine. The method applies solid-phase extraction with WCX cartridges for plasma and MM2 cartridges for urine, prior to analysis by LC/ESI-MS(n). The efficiency of extraction combined with the sensitivity and the selectivity of MS(n) allows for the quantification of ipratropium cation at picogram per milliliter levels. The analytical capabilities of the method have been successfully checked by the quantitative analysis of ipratr...
Ginther OJ, Beg MA.The temporal associations of cortisol, estradiol-17β, and oxytocin with pulses of PGFM at the common hour of transition between preluteolysis and luteolysis was studied in plasma from hourly blood samples in mares (n=8). The transitional hour was determined from progesterone concentrations and occurred between 2PM and 2AM in all mares. Pulses of PGFM were grouped into those occurring at the last pulse of preluteolysis (preluteolytic pulse), at the hour of transition (transitional), and during luteolysis (luteolytic). The preluteolytic PGFM pulse (45±16pg/ml at peak) and transitional pulse (4...
Blatti S, Overesch G, Gerber V, Frey J, Hüssy D.A retrospective, cross-sectional study was conducted to determine the leptospiral seroprevalence in clinically healthy horses in Switzerland. A representative sample of 615 horse sera was examined by microscopic agglutination test for the presence of antibodies against 15 Leptospira spp. serovars. In total, 58.5 % (n = 360) of the horses were positive for one or more of the antigens analysed, with 20.3 % of them showing titres >= 400. The most prevalent serovar was Pyrogenes (22.6 %), followed by serovars Canicola (22.1 %) and Australis (19.2 %). Older horses, mares, ponies and ani...
Pronost S, Legrand L, Pitel PH, Wegge B, Lissens J, Freymuth F, Richard E, Fortier G.Equid herpesvirus 1 (EHV-1)-associated myeloencephalopathy (EHM) is a disease affecting the central nervous system of horses. Despite the constantly increasing interest about this syndrome, epidemiological data are limited especially when related to the description of large outbreaks. The aim of this article is to describe clinical, virological and molecular data obtained throughout a severe outbreak of EHM, with emphasis on laboratory diagnostic methods. The epidemic disease concerned a riding school in France where 7/66 horses aged 12-22 years developed signs of neurological disease in July ...
D'Arcy-Moskwa E, Weston L, Noble GN, Raidal SL.Mucosal integrity may be objectively assessed by determination of the absorption of exogenous substances such as sucrose. Gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC/MS) have been reported for the accurate quantification of low concentrations of sucrose in serum. LC/MS offered the advantage of high sensitivity and mass selectivity without the need for extensive sample derivatization required for GC/MS methods. However, the high polarity and non-volatile nature of the sucrose molecule renders LC/MS techniques challenging. Previously published rep...
Hagen J, Hartnett C, Houchins JP, Giguère S, Kalyuzhny AE.Human and mouse immune system cells are the most frequently used specimens in ELISPOT assays. In an effect to expand the application of ELISPOT assay to other species, we developed matched antibody pairs for ready-to-use kits designed for studying the frequency of equine IFNγ- and IL-4-secreting peripheral blood mononuclear cells (PBMCs). Equine PBMCs were stimulated with either concanavalin A (Con A) or calcium ionomycin mixed with phorbol 12-myristate 13-acetate (CaI + PMA). We found that Con A, in general, had a more profound stimulating effect than CaI + PMA on IL-4 secretion, whereas bot...
Medana C, Calza P, Giancotti V, Dal Bello F, Pasello E, Montana M, Baiocchi C.Two horses were treated with sildenafil, and its metabolic products were sought in both urine and plasma samples. Prior to this, a simulative laboratory study had been done using a photocatalytic process, to identify all possible main and secondary transformation products, in a clean matrix; these were then sought in the biological samples. The transformation of sildenafil and the formation of intermediate products were evaluated adopting titanium dioxide as photocatalyst. Several products were formed and characterized using the HPLC/HRMS(n) technique. The main intermediates identified in thes...
Wauters J, Franck T, Pille F, Martens A, Demeyere K, Sys S, Serteyn D, Gasthuys F, Meyer E.Myeloperoxidase (MPO) is a protein of interest due to its involvement in equine pathologies. Until now, results in equine diagnostic research were achieved through extracellular MPO detection. However, studying the cellular MPO content in neutrophils has revealed important insights in human diseases. This study aimed to develop a technique for the specific detection of MPO on the single cell level defining a flow cytometric protocol for the detection of both equine surface-bound and cellular MPO. Both indirect and direct labeling techniques are described which include the comparison of two sec...
Appolonova SA, Baranov PA, Mesonzhnik NV, Brazhnikova DO, Rodchenkov GM.A method is described for the determination of mesocarb abuse in equestrian sport by combining gradient liquid chromatography and electrospray ionization tandem mass spectrometry. Mesocarb was administrated orally to two horses at a dose of 50 µg/kg. Urine samples were collected up to 120 h post administration. Hydrolyzed and conjugated urine fractions were handled using liquid-liquid extraction (LLE). The identity of the parent drug and metabolites was confirmed using liquid chromatography combined with tandem mass spectrometry (MS/MS). Mesocarb and seven metabolites were detected in horse...
Scheffer EG, Venter GJ, Labuschagne K, Page PC, Mullens BA, MacLachlan NJ, Osterrieder N, Guthrie AJ.Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of a variety of pathogens including African horse sickness virus (AHSV), a member of the family Reoviridae, genus Orbivirus. AHSV causes African horse sickness (AHS), an endemic disease of equids with an extremely high mortality rate in horses in sub-Saharan Africa. Culicoides (Avaritia) imicola Kieffer is considered to be the principal vector of AHSV and is the dominant Culicoides species in South Africa. Due to the global distribution of Culicoides vectors, there is a potential risk of AHS spreading from endemic areas to areas t...
Azab W, El-Sheikh A, Abdel-Gawad A.Equine herpesvirus 4 (EHV-4) is an important pathogen that causes respiratory tract disease in horse populations worldwide. Glycoprotein G (gG) homologs have been identified in several alphaherpesviruses as minor non-essential membrane-anchored glycoproteins. In this study, EHV-4 gG deletion mutant has been generated by using bacterial artificial chromosome technology to investigate the role of gG in viral pathogenesis. Our findings reported here revealed no significant difference between parental EHV-4 and gG-negative strain in their replication cycle in cell culture. Furthermore, virus titer...
Deschene K, Céleste C, Boerboom D, Theoret CL.Exuberant granulation tissue (EGT), a fibrotic healing disorder resembling the human keloid, occurs almost exclusively in limb wounds of horses and may be caused in part by a relative state of hypoxia within the wound. Objective: The objectives of this study were therefore to (1) assess the effects of hypoxia on equine dermal fibroblast (EDF) proliferation and apoptosis, (2) study the effects of hypoxia on the expression of key extracellular matrix (ECM) associated proteins and determine if such effects are dependent on hypoxia-inducible factor (HIF), and (3) determine if EDFs from the body or...
Barrey E, Jayr L, Mucher E, Gospodnetic S, Joly F, Benech P, Alibert O, Gidrol X, Mata X, Vaiman A, Guérin G.Recurrent exertional rhabdomyolysis (RER) is frequently observed in race horses like trotters. Some predisposing genetic factors have been described in epidemiological studies. However, the exact aetiology is still unknown. A calcium homeostasis disruption was suspected in previous experimental studies, and we suggested that a transcriptome analysis of RER muscles would be a possible way to investigate the pathway disorder. The purpose of this study was to compare the gene expression profile of RER vs. control muscles in the French Trotter to determine any metabolic or structural disruption. T...
Böer U, Lohrenz A, Klingenberg M, Pich A, Haverich A, Wilhelmi M.Decellularized equine carotid arteries (dEAC) may represent a reasonable alternative to alloplastic materials in vascular replacement therapy. Acellularity of the matrix is standardly evaluated by DNA quantification what however may not record sufficiently the degree of matrix immunogenicity. Thus, our aim was to analyze dEAC with a low DNA content for residual cellular proteins. A detergent-based decellularization protocol including endonuclease treatment resulted in dEAC with 0.6 ± 0.15 ng DNA/mg dry weight representing 0.33 ± 0.14% of native tissue DNA content. In contrast, when matrices ...
Stege PW, Lapierre AV, Martinez LD, Messina GA, Sombra LL.In this study we developed an interesting alternative to HPLC-mass spectrometry for the quantification of seven important drugs of abuse in racehorses. The procedure proposed in this work is a combination of single-drop microextraction (SDME) and an open tubular capillary electrochromatography (OT-CEC) using multi-wall carbon nanotubes (MWCTs) immobilized into a fused-silica capillary as a stationary phase. The SDME showed to be a powerful tool for extraction/preconcentration of the seven drugs analyzed in the study, showing an enrichment factor between 38- and 102-fold depending on the drug. ...
Corbin LJ, Blott SC, Swinburne JE, Vaudin M, Bishop SC, Woolliams JA.We have used linkage disequilibrium (LD) to identify single nucleotide polymorphisms (SNPs) on the Illumina Equine SNP50 BeadChip, which may be incorrectly positioned on the genome map. A total of 1201 Thoroughbred horses were genotyped using the Illumina Equine SNP50 BeadChip. LD was evaluated in a pairwise fashion between all autosomal SNPs, both within and across chromosomes. Filters were then applied to the data, firstly to identify SNPs that may have been mapped to the wrong chromosome and secondly to identify SNPs that may have been incorrectly positioned within chromosomes. We identifie...
Fenwick SJ, Scarth JP.Within horseracing, the detection of prohibited substance doping often requires urine analysis; hence, it is necessary to understand the metabolism of the drugs in question. Here, the previously unknown equine metabolism of eight sedatives is reported in order to provide information on target metabolites for use in doping control. Phase I metabolite information was provided by incubation with equine liver S9 fraction. In vitro techniques were chosen in order to reduce the ethical and financial issues surrounding the study of so many compounds, none of which are licensed for use in horses in th...
Wassmuth AK, Riond B, Hofmann-Lehmann R, Lutz H.The Mythic 18 is a fully automated hematology bench-top analyzer using impedance technology for a complete blood cell count (CBC) and a 3-part white blood cell count (WBC) differential. The purpose of the current study was to evaluate the Mythic for assessment of agreement, precision, linearity, carry-over, stability, and usability under practice conditions. Ethylenediamine tetra-acetic acid-blood samples from 122 dogs, 140 cats, and 123 horses were analyzed with the Mythic and reference methods (Sysmex XT-2000iV, manual hematocrit, and microscopic WBC differentiation). Pearson's coefficient o...
Desta B, Maldonado G, Reid H, Puschner B, Maxwell J, Agasan A, Humphreys L, Holt T.Just prior to an international polo event, 21 horses from one team exhibited clinical signs of central nervous system disturbance, hyperexcitability, sweating, ataxia, tachycardia, dyspnea, pyrexia, and rapid death. The suspected cause of this peracute onset of illness and death included intentional contamination of feed or iatrogenic administration of performance-enhancing drugs resulting in a severe adverse reaction. Six horses were submitted to the Bronson Animal Disease Diagnostic Laboratory for necropsy and toxicological examination. The clinical signs and sudden death, the similarity to ...
Artiushin S, Tong Y, Timoney J, Lemieux B, Schlegel A, Kong H.A simple and portable assay for detection of Streptococcus equi subspecies equi has been developed based on amplification of S. equi-specific sequence using a thermophilic helicase-dependent reaction followed by visual detection of the amplicon in a disposable lateral flow cassette. An experimental kit (IsoAmp™ SE) was evaluated. Analytical sensitivity was 50 copies of S. equi genomic DNA per reaction. The IsoAmp SE assay had 100% specificity when applied to nasal swabs and washes. The assay was more sensitive than culture but less sensitive than nested polymerase chain reaction (PCR). The t...
Dunkel B, Bolt DM, Smith RK, Cunningham FM.Platelet-rich plasma (PRP) is increasingly used for treatment of orthopaedic injuries. However, the effects of different stimuli on the release pattern of regenerative and proinflammatory factors from equine platelets are largely unknown and an optimal treatment protocol remains to be established. Objective: The aim of this study was to identify a stimulus that enhanced release of histopromotive factors (platelet-derived growth factor BB [PDGF] and transforming growth factor 1β[TGF]) without causing concurrent release of a proinflammatory mediator (CCL5). Methods: Washed platelets were prepar...
Casella S, Giudice E, Giannetto C, Marafioti S, Piccione G.The purpose of this study was to evaluate in vitro the effects of hydrocortisone and aminophylline on adenosine diphosphate (ADP)-induced platelet aggregation in horses. Blood samples from 30 healthy Thoroughbred horses were collected by via jugular venipuncture to assess platelet aggregation. Platelet-rich and platelet-poor plasma were prepared from all samples by centrifugation and divided into three different aliquots. In the first aliquot, platelet aggregation was measured after platelet activation with 1 µM and 0.5 µM ADP (Group A). In the other two aliquots, the effect of a 10 min prei...
Kuroda T, Minamijima Y, Niwa H, Tamura N, Mita H, Fukuda K, Kaimachi M, Suzuki Y, Enoki Y, Taguchi K, Matsumoto K, Toutain PL, Bousquet-Melou A....First-generation cephalosporins have good activity against gram-positive bacteria and are extensively used in horses. There are few reports of pharmacokinetics and pharmacodynamics (PK/PD) analysis of cephalosporins in horses. Objective: To optimise the dosages of the two first-generation cephalosporins cephalothin (CET) and cefazolin (CEZ) in horses using PK/PD concepts. Methods: Experimental study with single administration. Methods: Drug plasma concentrations following a single intravenous (i.v.) administration of 22 mg/kg bodyweight (bwt) CET in 12 horses and of 10 mg/kg bwt CEZ in six h...
Virmani N, Bera BC, Shanumugasundaram K, Singh BK, Gulati BR, Singh RK, Vaid RK.India faced an epizootic of equine influenza in 2008-2009. The isolated viruses were typed as H3N8 and grouped with the clade 2 viruses of Florida sublineage on the basis of haemagglutinin (HA) gene sequence analysis. This report describes the genetic analysis and selection pressure of matrix (M) and non-structural 1 (NS1) genes of the Indian isolates. All isolates shared 98.41% and 99.54% homology with other clade 2 viruses of Asian origin for M1 and M2 amino acid (aa) sequences, respectively. There were 3 and 4 unique aa residue changes respectively in M1 and M2 proteins in all Asian isolate...
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...
Covaleda L, Gno BT, Fuller FJ, Payne SL.The macrophage-tropic lentivirus, equine infectious anemia virus (EIAV), encodes the small auxiliary protein S2 from a short open reading frame that overlaps the amino terminus of env EIAV S2 is dispensable for virus replication in cultured cells but is required for disease production. S2 is approximately 7 kDa and has no overall amino acid sequence homology to other cellular or viral proteins. Therefore it is likely that S2 plays a role as an adaptor protein. To further investigate S2 function we performed a yeast-2-hybrid screen to identify cellular proteins that interact with EIAV S2. The s...
Stone-Marschat M, Carville A, Skowronek A, Laegreid WW.Reverse transcription-PCR (RT-PCR) was used to detect African horse sickness virus (AHSV). A single primer pair which amplified a 423-bp fragment of the S8 gene which encodes the NS2 protein of AHSV was identified. Amplification of this fragment from all nine serotypes of AHSV was achieved with these primers. Between 10(1) and 10(2) copies of AHSV genomic double-stranded RNA could be detected by RT-PCR followed by agarose gel electrophoresis and ethidium bromide staining. Application of RT-PCR to blood samples from AHSV-infected horses resulted in earlier detection of viremia than virus isolat...
Wadsworth AB, Kirkbride MB.Horses immunized to Type I pneumococci developed serum, 0.1 cc. of which protected against 0.5 cc. of a virulent culture, 0.000001 cc. of which killed mice in less than 40 hours. Protective tests of serum from horses immunized to Type II organisms varied, 0.1 cc. protecting, however, in certain instances against 0.1 and 0.01 cc. of virulent homologous culture. Types I and II sera obtained in our experiments with culture sediment and whole culture did not vary markedly for a given type and corresponded closely in their protective titer with samples of sera received from The Rockefeller Institut...
Alvarez I, Gutierrez G, Ostlund E, Barrandeguy M, Trono K.We analyzed the performance of a single-band Western blot (WB) test using recombinant p26 (rp26) capsid protein of equine infectious anemia virus. According to the results obtained, the rp26 WB test is a reliable confirmatory diagnostic tool to be used as a complementary test after an enzyme-linked immunosorbent assay or agar gel immunodiffusion test yielding doubtful results.
Myer YP, Kumar S.The ascorbate reduction of horse heart ferricytochrome c in 0.05 M phosphate + 0.25 M sodium sulfate, at pH 7.3, as a function of temperature, 12-36 degrees C, and at alkaline pH 8.4 using stopped flow technique has been examined. The data have been analyzed in terms of a two-step mechanism, binding followed by reduction (Myer, Y.P., Thallam, K.K., and Pande, A. (1980) J. Biol. Chem. 255, 9666-9673). At neutral pH and up to about 26 degrees C, the first order reduction constant is independent of temperature, i.e. with zero or near-zero activation energy. At higher temperatures, it becomes temp...
Salas Y, Márquez A, Canelón J, Perazzo Y, Colmenárez V, López JA.Pythium insidiosum is a pathogenic oomycete known since 1890 that causes pythiosis in mammals. In this report, seven P. insidiosum isolates were recovered from Venezuelan horses and were characterized. The strains were recovered from biopsied tissues and kunkers collected from granulomatous masses located on the hind limb and from a nodular lesion in the left upper eyelid, which decrease the ability of the horses to be used for working purposes. The methods used to identify P. insidiosum isolates were based on the production of sporangia and zoospores, histopathology and PCR assay. To further ...
Chambers TM, Reedy SE.Serologic tests for equine influenza virus (EIV) antibodies are used for many purposes, including retrospective diagnosis, subtyping of virus isolates, antigenic comparison of different virus strains, and measurement of immune responses to EIV vaccines. The hemagglutination-inhibition (HI), single radial hemolysis (SRH), and serum micro-neutralization tests are the most widely used for these purposes and are described here. The presence of inhibitors of hemagglutination in equine serum complicates interpretation of HI assay results, and there are alternative protocols (receptor-destroying enzy...
Stefanini S, Chiancone E, Vecchini P, Antonini E.Iron uptake and micelle formation in ferritin and apoferritin have been followed both spectrophotometrically and by means of sedimentation velocity experiments. Information was thus obtained on the molecular weight distribution of the reconstitution product. To achieve incorporation 'native' ferritin (whole ferritin as purified from horse spleen), 'native' apoferritin (apoferritin prepared by fractionation of ferritin preparations) and 'reduced' apoferritin (apoferritin prepared by reduction of ferritin by dithionite or ascorbic acid) have been incubated with ferrous salts in the presence of o...
Karimi A, Mosavari N.Burkholderia mallei, the etiologic agent of the disease known as glanders. Clinical and bacteriological diagnosis of glanders is difficult in the early stages of the disease. Currently, mallein (allergic hypersensitivity test) is used for the diagnosis of glanders. The mallein test requires an experienced laboratory person and lasts 48 h. Therefore, in order to quickly diagnose the disease, especially in areas (such as the borders of the country) that cannot be kept animals, new methods should be used to identify the disease. The Rose Bengal is a serological diagnostic test and has been recom...
Ben David A, Barnea A, Torgeman A, Diamant E, Dor E, Schwartz A, Rosen O, Caspi N, Saraf M, Lerer E, Adar Y, Lupo E, Toister E, Zichel R.Botulism is a paralytic disease caused by botulinum neurotoxins (BoNTs). Equine antitoxin is currently the standard therapy for botulism in human. The preparation of equine antitoxin relies on the immunization of horses with botulinum toxoid, which suffers from low yield and safety limitations. The Hc fragment of BoNTs was suggested to be a potent antibotulinum subunit vaccine. The current study presents a comparative evaluation of equine-based toxoid-derived antitoxin (TDA) and subunit-derived antitoxin (SDA). The potency of recombinant Hc/A, Hc/B, and Hc/E in mice was similar to that of toxo...
Bailey E, Reid RC, Skow LC, Mathiason K, Lear TL, McGuire TC.Equine combined immunodeficiency disease (CID) is caused by homozygosity for an autosomal recessive gene. To identify linked markers for the disease, we studied a family segregating for the equine CID gene. A stallion and 19 of his CID-affected offspring were tested for marker segregation at 23 microsatellite DNA loci. His CID-affected offspring inherited only one of his two alleles at the HTG8 and HTG4 loci, namely HTG8-186 and HTG4-124, respectively. Lod scores for linkage to the CID gene using a theta of 0.01 were 5.34 for HTG8 and 2.37 for HTG4. The apparent genotypes also suggested linkag...
Latif ZA, Fletcher MA.Immunochemical comparisons were made of the reactivity of membrane glycoproteins from horse, bovine, sheep and goat erythrocytes with heterophile antibodies of infectious mononucleosis. The four receptors were tested as competitive inhibitors of a sandwich-type solid-phase radioimmunoassay and of agglutination of glycoprotein-latex reagents by infectious mononucleosis serum. The results of this study showed that the bovine glycoprotein had a superior reactivity with this heterophile antibody system and sheep erythrocyte glycoprotein was the least reactive. The latter had negligible ability to ...
Schnabel CL, Wagner S, Wagner B, Durán MC, Babasyan S, Nolte I, Pfarrer C, Feige K, Murua Escobar H, Cavalleri JM.Research on equine cytokines is often performed by analyses of mRNA. For many equine cytokines an analysis on the actual protein level is limited by the availability of antibodies against the targeted cytokines. Generation of new antibodies is ongoing but time consuming. Thus, testing the reactivity of commercially available antibodies for cross-reactivity with equine cytokines is of particular interest. Fifteen monoclonal antibodies against IL-1β, IL-6, IL-8, IL-12, IL-18 and Granulocyte Macrophage Colony stimulating factor (GM-CSF) of different species were evaluated for reactivity with the...
Yamanaka T, Tsujimura K, Kondo T, Matsumura T.In this study, we evaluated whether five rapid antigen detection kits for human influenza could be used for the diagnosis of equine influenza (EI). Limiting dilution analyses showed that Directigen Flu A+B and ESPLINE INFLUENZA A&B-N had the highest sensitivities to equine-2 influenza viruses (EIVs) among the kits investigated. From the results of virus detection in nasal swabs taken from horses infected with EIV, these two kits could produce positive results in reasonable agreement with those obtained by virus isolation or RT-PCR, suggesting that these kits could be useful for rapid diagn...
Cohen ND, Neibergs HL, Wallis DE, Simpson RB, McGruder ED, Hargis BM.Members of the genus Salmonella were identified in feces from horses, using the polymerase chain reaction (PCR) and genus-specific oligonucleotide primers. Feces from healthy horses were determined to be culture-negative for Salmonella spp. Fecal samples were inoculated with known numbers of colony-forming units (CFU) of S anatum, S derby, S enteritidis, S heidelberg, S newport, and S typhimurium. The DNA was extracted from fecal samples and amplified by PCR, using genus-specific primers. Sensitivity of the assay extended to 10(3) CFU of Salmonella sp/g of feces; sensitivity of microbiologic c...
Bailey MT, Troedsson MH, Wheato JE.The hormone-producing equine granulosa cell tumor (GCT) may secrete high levels of inhibin. Measurement of inhibin concentrations may be useful in the diagnosis and conformation of mares with GCT. Inhibin may be measured using RIA, which recognizes dimeric alphabetaA-inhibin as well as the monomeric (free) inhibin alpha-subunit, or using a two-site immunoradiometric assay (IRMA) specific for alphabetaA-inhibin. The objective of this study was to examine concurrent relationships among alpha-inhibin (as measured using RIA), alphabetaA-inhibin (as measured using IRMA), and other hormones (testost...
Stojiljkovic N, Paris A, Garcia P, Popot MA, Bonnaire Y, Tabet JC, Junot C.Horse urine is the medium of choice for the implementation of metabolomic approaches aimed at improving horse doping control. However, drug analysis in this biofluid is a challenging task due to the presence of large amounts of interfering compounds. METHODOLOGY & RESULTS: A comparative study of sample preparation has been conducted to evaluate five sample-preparation methods, namely acetonitrile precipitation, proteinase K hydrolysis, membrane filtration and sample dilution with water by factors of five and 20, for metabolome analysis using liquid chromatography coupled to high resolution...
Ji Y, Guo W, Zhao L, Li H, Lu G, Wang Z, Wang G, Liu C, Xiang W.An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed for the detection of the equine influenza virus (EIV), employing monoclonal and polyclonal antibodies against the A/equine/Xingjiang/2007 (H3N8) nucleoprotein (NP). Immunoglobulin G antibodies were purified and used as capture or detector antibodies. The specificity of the optimized AC-ELISA was evaluated using EIV, equine herpesvirus 1 (EHV-1), equine herpesvirus 4 (EHV-4), equine arteritis virus (EAV) and Japanese encephalitis virus (JEV), resulting in only EIV specimens yielding a strong signal. A minimal concentr...
Timms M, Hall N, Levina V, Vine J, Steel R.The growth hormone releasing peptides (GHRPs) hexarelin, ipamorelin, alexamorelin, GHRP-1, GHRP-2, GHRP-4, GHRP-5, and GHRP-6 are all synthetic met-enkephalin analogues that include unnatural D-amino acids. They were designed specifically for their ability to stimulate growth hormone release and may serve as performance enhancing drugs. To regulate the use of these peptides within the horse racing industry and by human athletes, a method is presented for the extraction, derivatization, and detection of GHRPs from equine and human urine. This method takes advantage of a highly specific solid-ph...
Jiang Z, Haughan J, Moss KL, Stefanovski D, Ortved KF, Robinson MA.Gene therapy is currently prohibited in human and equine athletes and novel analytical methods are needed for its detection. Most in vivo products use non-integrating, recombinant viral vectors derived from adeno-associated virus (AAV) to deliver transgenes into cells, where they are transcribed and translated into functional proteins. Although the majority of wild-type AAV (WTAAV) DNA is removed from recombinant AAV (rAAV) vectors, some sequences are conserved. The goal of this study was to develop a quantitative polymerase chain reaction (QPCR) screening test targeting conserved AAV sequence...
Mankoc S, Hostnik P, Grom J, Toplak I, Klobucar I, Kosec M, Barlic-Maganja D.In the present study, a new one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) strategy with minor-groove-binder (MGB) technology for the detection of EAV from 40 semen samples of Slovenian carrier stallions was tested. A novel MGB probe (EAVMGBpr) and a reverse primer (EAV-R) based on the multiple sequence alignment of 49 different EAV strain sequences of the highly conserved ORF7 (nucleocapsid gene) were designed. The performance of the assay was compared with different molecular detection methods. Three different primer pairs targeting the ORF1b and ORF7 were used, ...
Frean SP, Lees P.To investigate effects of the anti-arthritic agents hyaluronan and polysulfated glycosaminoglycan (PSGAG) on inflammatory metabolism in cultured equine synoviocytes. Methods: Synoviocytes cultured from samples obtained from the metacarpophalangeal joints of 4 horses. Methods: Equine synoviocytes were grown in monolayer culture. Synoviocytes were stimulated with lipopolysaccharide (LPS) and simultaneously treated with various concentrations of hyaluronan or PSGAG for 48 hours. Three hyaluronan preparations were compared. Prostaglandin E2 (PGE2) concentrations in culture medium were measured, us...
Atherton JG, Pitt TL.Pseudomonas aeruginosa isolates from equine clinical material were categorised according to their serotype and phage type. Epidemiological evidence showed that serotypes 02a, 03, 04, 06, 09 and 010 were the cause of genital and non-genital infections; somatic type 03 accounted for 50 per cent of isolates. The laboratory tests used were of no value in predicting whether or not a particular isolate was likely to be a venereal pathogen, but all the serotypes encountered had the potential to be pathogenic, given a favourable environment in which to multiply.
Alvarez I, Cipolini F, Wigdorovitz A, Trono K, Barrandeguy ME.The most used and reliable indicator of Equine infectious anemia virus (EIAV) infection is the detection of its specific antibodies in horse serum. In the present study, the performance of two commercial ELISA tests for the detection of EIAV antibodies as well as the potential advantages of their use as an EIAV infection screening tool were evaluated in 302 horse serum samples. Both ELISA assays showed 100% diagnostic sensitivity, and 92.3-94.3% diagnostic specificity. Discordant results were analyzed by immunoblot. The results showed that both ELISA tests are very efficient at detecting EIAV ...
Hales EN, Habib H, Favro G, Katzman S, Sakai RR, Marquardt S, Bordbari MH, Ming-Whitfield B, Peterson J, Dahlgren AR, Rivas V, Ramirez CA, Peng S....Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) is an inherited neurodegenerative disorder associated with a vitamin E deficiency within the first year of life. Vitamin E consists of 8 isoforms metabolized by the CYP4F2 enzyme. No antemortem diagnostic test currently exists for eNAD/EDM. Objective: Based on the association of α-tocopherol deficiency with the development of eNAD/EDM, we hypothesized that the rate of α-tocopherol, but not γ-tocopherol or tocotrienol metabolism, would be increased in eNAD/EDM-affected horses. Methods: Vitamin E metabolism: Proof...
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...
Tomczuk K, Kostro K, Grzybek M, Szczepaniak K, Studzińska M, Demkowska-Kutrzepa M, Roczeń-Karczmarz M.For this study, 724 gastrointestinal tracts of slaughter horses were investigated to determine the prevalence, intensity of Anoplocephala perfoliata and tapeworm development stages over the second, third and fourth quarter of 2012 and the first quarter of 2013. For each positive horse, faecal samples were collected from the rectum or small colon for coproscopic examinations. The samples were analysed using dedicated modified sedimentation-flotation methods. In total, 52 horses were infected with A. perfoliata in the course of the study, with an overall prevalence of 7.2 %. The prevalence chang...
