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.
AZZONE GF.In a series of model reactions, it is shown that residues
of ~-aminoacids may be inserted by a particular rearrangement into certain carboxyl or carbonylamido
groups. Repeated insertion results in the formation of
a peptide derivative. It is concluded that natural peptides or proteins must not necessarily be formed by head
to tail combination of aminoacids, Other implications of
the new principle are discussed.
Haysom LZ, Lee-Fowler TM, Spangler EA.Storage and temperature significantly impact bronchoalveolar lavage fluid (BALF) analysis, and shipment of samples to diagnostic laboratories is often necessary. Alternative sample preparation methods could limit storage and temperature effects. Objective: This study aimed to determine if airway wash samples that were fixed in formalin after being embedded in Histogel or Gelfoam gave comparable results to fresh cytocentrifuged or sediment smear preparations for the evaluation of cell morphology. Methods: Eleven bronchoalveolar lavage and 3 transtracheal wash fluids were available, including 8 ...
von Samson-Himmelstjerna G, Janssen IJI, Ramünke S, Goday C, Borges FA, Koudela B, Niedźwiedź A, Tomczuk K, Studzińska MB, Kornas S, Krücken J.Equines were over decades considered to be infected by two morphologically virtually indistinguishable ascarid species, Parascaris univalens and Parascaris equorum. Reliable species discrimination is only possible using enzyme isoelectric focussing and karyotyping with P. univalens having one and P. equorum two chromosome pairs. However, presumably the complexity of both methods prevented their routine use in nearly all previous studies about prevalence and drug resistance of Parascaris spp. These have barely been performed on the species level although most studies stated presence of one or t...
Rotkina AS, Pronina IV, Lazarev VN, Akhaev DN, Baskova IP.The purpose. Identifying the capacity of the medicinal leech novel original recombinant thrombolytic preparation Destabilase-Lysozyme-2 to inhibit the blood platelet aggregation. Gene of destabilase-lysozyme. ds2 (mlDL-Ds2 ), was cloned in E.coli cells. Recombinant protein was isolated in denaturing conditions using metal-chelate chromatography followed by denaturation of the polypeptide by rapid dilution in exact accordance with the procedure described by Kurdyumov A.S. et al. ( 2016, Russian Journal of Bioorganic Chemistry, v.42, s. 42-52). Blood was collected from the jugular vein of 18 hor...
van Gennip RGP, van de Water SGP, Potgieter CA, van Rijn PA.The Reoviridae family consists of nonenveloped multilayered viruses with a double-stranded RNA genome consisting of 9 to 12 genome segments. The Orbivirus genus of the Reoviridae family contains African horse sickness virus (AHSV), bluetongue virus, and epizootic hemorrhagic disease virus, which cause notifiable diseases and are spread by biting Culicoides species. Here, we used reverse genetics for AHSV to study the role of outer capsid protein VP2, encoded by genome segment 2 (Seg-2). Expansion of a previously found deletion in Seg-2 indicates that structural protein VP2 of AHSV is not essen...
Bachmann V, von Salis B, Fürst A.The goal of this study was to describe the development of equine drug testing in horses in Switzerland. This was achieved through evaluation of a film made by the Institute of Forensic Medicine at the University of Basel entitled 'Doping von Rennpferden' [Doping of Race Horses], toxicological detection, 1962', the analysis of doping test results of the Swiss Equestrian Federation and by interviewing individuals of various professions who were involved in equine drug testing at the time. The study compares early and modern methods of drug testing and highlights the changes in the attitude of eq...
Wells M, Gaffin SL.Passive immunotherapy with anti-lipopolysaccharide hyperimmune equine plasma (Anti-LPS) is effective in treating experimental Gram-negative bacterial infections. The bactericidal activity of anti-LPS towards five different Pseudomonas species, including two multiresistant Pseudomonas aeruginosa isolates was tested here, as well as the ability of anti-LPS to inhibit the quantitative chromogenic limulus amoebocyte lysate (LAL) assay. Anti-LPS caused a mean reduction of 84.4 +/- 3.2% (P less than 0.001) in the number of colony forming units (cfu) of all isolates, whereas saline and complement ina...
Xiao C, Liu Y, Jiang Y, Magoffin DE, Guo H, Xuan H, Wang G, Wang LF, Tu C.Four monoclonal antibodies (mAbs) were generated by immunizing BALB/C mice with recombinant nucleocapsid protein (N) of Nipah virus (NiV) and Hendra virus (HeV) expressed in E. coli. Two mAbs each were obtained for the HeV N and NiV N, respectively. All four mAbs displayed specific reactivity with the recombinant N proteins of both viruses by western blot, which was further confirmed by immunofluorescent antibody assay using fixed insect cells infected with recombinant baculoviruses expressing either the HeV or NiV N protein. Epitope mapping using a 12-mer random peptide phage display library ...
Hu WG, Chau D, Wong C, Masri SA, Fulton RE, Nagata LP.The genes encoding envelope proteins E1 and E2 of western equine encephalitis virus (WEEV) were respectively cloned into a prokaryotic T7 RNA polymerase-regulated expression vector. The recombinant C-terminal 6xHis-tagged WEEV E1 and E2 were expressed in bacteria as inclusion bodies that were subsequently solubilized with 8M urea, purified by immobilized metal ion affinity chromatography and finally refolded using an arginine system. The purified 6xHis-tagged proteins showed 50kDa bands as revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, consistent with the expected size...
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...
Metenier L, Grosclaude J, Meriaux JC.The chief application of blood typing in domestic animals is in the verification of parentage. However, the acquisition of good standardized reagents in sufficient quantity remains an obstacle for the development of this work. The production of monoclonal antibodies directed against blood group determinants offers an attractive means of improving both the quality and quantity of serological reagents, and could facilitate the definition of new specificities. Fusions between a mouse myeloma line and splenocytes from mice immunized with horse red cells have resulted in four hybridomas producing a...
Terpstra AH, Sanchez-Muniz FJ, West CE, Woodward CJ.1. By means of density gradient ultracentrifugation, the density profile of the serum lipoproteins was studied in 14 species of domestic and laboratory animals: the pig, chicken, rhesus monkey, rabbit, dog, horse, sheep, cat, mouse, goat, cow, guinea-pig, trout and rat. 2. The concentration of cholesterol in whole serum and the lipoprotein fractions of these animal species was also determined. 3. There were large differences in the density profile of the serum lipoproteins among the various animals studied and the results indicate that the density limits employed for human serum lipoproteins a...
Ladrón N, Fernández M, Agüero J, González Zörn B, Vázquez-Boland JA, Navas J.The actinomycete Rhodococcus equi is an important pathogen of horses and an emerging opportunistic pathogen of humans. Identification of R. equi by classical bacteriological techniques is sometimes difficult, and misclassification of an isolate is not uncommon. We report here on a specific PCR assay for the rapid and reliable identification of R. equi. It is based on the amplification of a fragment of the choE gene encoding cholesterol oxidase. The choE-based PCR was assessed by using a panel of strains comprising 132 isolates from different sources and of different geographical origins, all i...
Theodorakis JL, Armes LG, Margoliash E.beta-Thiopropionyl derivatives of horse cytochrome c singly modified at each of 18 different lysine epsilon-amino groups have been prepared using sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate and purified to homogeneity by high-pressure liquid chromatography. These derivatives were characterized by determination of: (i) the location of the modification; (ii) reduction potentials; (iii) visible and NMR spectra: and by (iv) measurement of electron transfer activity with cytochrome-c oxidase. No significant changes in structure were indicated, except for the ferric forms of the deri...
Heo EJ, Lee HS, Jeoung HY, Ko HR, Kweon CH, Ko YJ.A recombinant glycoprotein (R-GP) of vesicular stomatitis New Jersey virus (VSV-NJ) was expressed in insect cells by a baculovirus system. Its utility as a diagnostic antigen in a blocking ELISA was investigated as an alternative to the current native GP extracted from VSV-NJ. With the cut-off value of 73% inhibition, the R-GP ELISA exhibited 99.1% specificity for naive sera from cattle and horses. It did not cross-react with VSV-Indiana (VSV-IN) positive sera and differentiated from foot-and-mouth disease and swine vesicular disease. Taken together, this is the first report that the R-GP has ...
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...
van Aken D, Zevenhoven-Dobbe J, Gorbalenya AE, Snijder EJ.The positive-stranded RNA genome of the arterivirus Equine arteritis virus (order Nidovirales) encodes the partially overlapping replicase polyproteins pp1a (1727 aa) and pp1ab (3175 aa). Previously, three viral proteinases were reported to cleave these large polyproteins into 12 non-structural proteins (nsps). The chymotrypsin-like viral main proteinase residing in nsp4 is responsible for eight of these cleavages. Processing of the C-terminal half of pp1a (the nsp3-8 region) was postulated to occur following either of two alternative proteolytic pathways (the 'major' and 'minor' pathways). He...
Kasza A, Korpula-Mastalerz R, Rose-John S, Dubin A.The horse leucocyte elastase inhibitor (HLEI), present in neutrophils, monocytes and bone marrow cells, is apparently a cytoplasmic protein which is not released from cells even in response to stimulation with lipopolysaccharide, phorbol ester, tumour necrosis factor alpha, interleukin-1 or elastin degradation products. Although no expression of the inhibitor was detected in neutrophils, both monocytes and bone marrow cells were efficient in its synthesis. Using a new expression vector pREST5d, recombinant inhibitor was produced in a large quantity in a soluble form, with a yield of 88 mg per ...
Franken P, Schotman JH.The activities and concentrations of a number of erythrocytic enzymes and intermediate products of erythrocyte metabolism were determined in twenty-one normal standard-bred horses which were studied clinically and biochemically. These studies showed that equine anaerobic glycolysis is characterized by a biochemical pattern similar to that observed in human PK deficiency. The greater sensitivity of equine haemoglobin to oxidants is attributable either to low stability of GSH, which may be due either to the low activity of GR or that of 6PGD as observed in the studies. In addition, the saturatio...
Zielińska-Dawidziak M, Błaszak R, Piasecka-Kwiatkowska D.The presented experiments show the model of expectation of equine spleen ferritin extraction in a new aqueous two-phase system which was formed by mixing polyethylene glycol (PEG) and hydroxyethyl starch (HES). The tendency of the protein to migrate in the analyzed systems was dependent on the concentrations of HES and PEG as well as PEG molecular weight. The highest concentration of ferritin in the top phase (rich in PEG) was recorded in the system composed of 6% PEG 3000 and 3% HES. The obtained concentration was 0.88 mg mL(-1). The lowest concentration was 0.42 mg mL(-1) in the system compo...
Guan M, Dai D, Li L, Wei J, Yang H, Li S, Zhang Y, Lin Y, Xiong S, Zhao Z.High levels of triacylglycerols (TGs) have been linked to cardiovascular disease and liver diseases. Comprehensively analyzing TGs is helpful to understand the TGs functions in these diseases. However, due to the existence of a large number of isomers TGs and the lack of commercial standards, precise analysis of individual triacylglycerol (TG) with specific fatty acid chain composition is full of challenge. In this work, ultra-performance liquid chromatography (UPLC) coupled with electrospray ionization (ESI) mass spectrometry (MS) were employed for comprehensive qualification and quantificati...
Wang CC, Hartmann-Fischbach P, Krueger TR, Wells TL, Feineman AR, Compton JC.Dermorphin and HYP(6) -dermorphin are hepta-peptides and natural opioids originally isolated from the skin of South American frogs. They are more potent than morphine but less likely to produce drug tolerance and addiction. These properties make them ideal candidates for the doping of racehorses to enhance performance during competition. Dermorphin was recently classified as a Class I drug by Racing Commissioners International (RCI), indicating that it is a banned substance in equine athletes. To enforce this ban, a fast and sensitive method was developed for dermorphin and HYP(6)-dermorphin a...
Oldenhof H, Schütze S, Wolkers WF, Sieme H.Sperm chromatin structure and condensation determine accessibility for damage, and hence success of fertilization and development. The aim of this study was to reveal characteristic spectral features coinciding with abnormal sperm chromatin packing (i.e., DNA-protein interactions) and decreased fertility, using Fourier transform infrared spectroscopy. Chromatin structure in spermatozoa obtained from different stallions was investigated. Furthermore, spermatozoa were exposed to oxidative stress, or treated with thiol-oxidizing and disulfide-reducing agents, to alter chromatin structure and pack...
Laroucau K, Lucia de Assis Santana V, Girault G, Martin B, Miranda da Silveira PP, Brasil Machado M, Joseph M, Wernery R, Wernery U, Zientara S....We present the first molecular characterisation based on MLVA and SNP analysis of a strain of Burkholderia mallei isolated from a mule found dead in Brazil in 2016.
Shivers CA, Liu IK.Sera and follicular fluid from mares previously determined as having antibodies to the zona pellucida were exposed to porcine ovarian oocytes before insemination with boar spermatozoa in vitro. There was a dramatic decline in the number of spermatozoa bound to zonae compared to treatments with sera and follicular fluid from mares negative for zona antibodies. These data suggest that antibodies reactive with the zona pellucida may be responsible for reduced or even complete infertility in some mares. In a group of 50 randomly selected mares tested for antizona antibodies, 2 pregnant mares were ...
In this study, a new sandwich-type immunoenzymatic assay, based on a molecularly imprinted polymer (MIP) as an artificial antibody (pseudo-ELISA), was developed for the determination of procalcitonin (PCT) in veterinary species. The quantification of PCT in human medicine represents the state of the art for the diagnosis of sepsis; instead the clinical studies on the relevance of PCT as a sepsis predictor in veterinary patients are few, likely due to the total absence of validated assays. MIPs have been widely used as antibody mimics for important applications, and MIP-based sandwich assays ha...
Macintyre AR, Dixon JB.To determine whether living hydatid tissue can, like hydatid fluid, regulate leukocyte growth, T-cell, B-cell, and macrophage lines were cocultured with protoscoleces of Echinococcus granulosus and their growth was compared with that of control cultures by thymidine uptake estimates and chemiluminescent assays of cell number. Protoscoleces supported mitosis of IL-1-deprived D10 T cells, but did not increase D10 count. The action of protoscoleces was affected by the species and organ of their origin and the length of time in culture. Unusually marked mitotic reaction, unaffected by parasite age...
Le Goff D, Hannan J, Maboundou JC, Ayrault-Jarrier M.Equine plasma lipoproteins were fractionated into VLDL, LDL-1, LDL-2 and HDL by density gradient ultracentrifugation. From each lipoprotein fraction, five apo C like peptides of approx. M(r) 1400, 10000, 9500, 9000 and 8000 were detected by SDS-polyacrylamide gel electrophoresis. After partial purification by Sephadex G-75, one fraction, showing a strong activation of lipoprotein lipase, was further purified by Mono Q anion exchange column. Two of the apo C like peptides (M(r) 10000 and 8000) activated the bovine milk lipoprotein lipase in vitro; only one (M(r) 9500) inhibited the lipolytic ac...
Eichhorn W.Infections and clinical diseases caused by equine 2 influenza A viruses are observed worldwide. The frequency of these outbreaks supports the hypothesis that antigenic variation of the surface proteins may play an important role. For the demonstration of these variations, monoclonal antibodies (Mabs) were prepared. They are directed against the hemagglutinin or the neuraminidase of the prototype strain a/eq/Miami/1/63. In hemagglutination-inhibition assays with Mabs two reaction patterns were observed: four Mabs inhibited 14 out of 17 strains tested. Another Mab recognized the hemagglutinin of...
Wu D, Murakami K, Liu N, Inoshima Y, Yokoyama T, Kokuho T, Inumaru S, Matsumura T, Kondo T, Nakano K, Sentsui H.The full-length equine interferon-gamma (eIFN-gamma) cDNA, including the secretion signal peptide coding region, was recloned into baculovirus transfer vector pAcYM1. This vector was co-transfected with Autographa californica nuclear polyhedrosis virus DNA or hybrid nuclear polyhedrosis virus DNA into Spodoptera frugiperda cells. The recombinant viruses, named AcEIFN-gamma and HyEIFN-gamma, were then recovered. Recombinant eIFN-gamma (reIFN-gamma) was accumulated in the culture fluid of the AcEIFN-gamma or HyEIFN-gamma infected Tricoplusia ni -derived cell line, BTI TN 5B1-4, and hemolymph of ...
Thomasy SM, Moeller BC, Stanley SD.To compare the density and binding characteristics of opioid receptor subtypes in horse, rat, and guinea pig cerebral cortex and cerebellum. Methods: Prospective receptor binding study. Methods: Whole brains were obtained from four neurologically normal adult horses during necropsy. Rat and guinea pig brains were obtained commercially. Methods: The cerebellum and cerebral cortex were dissected from each brain, and tissue homogenates prepared. A radioligand binding technique with the highly selective ligands [(3)H]-DAMGO, [(3)H]-U69593, and [(3)H]-DPDPE was used to identify the mu- (mu), kappa-...
