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.
White KL, Thomson DL, Wood TC.An indirect immunofluorescence assay was used to detect the presence of H-Y antigen on equine blastocysts. A total of 33 blastocyst stage horse embryos were collected 6 to 7 days post-ovulation by trans-cervical flush and were immediately evaluated for the presence of H-Y antigen. Additionally, 17 embryos, were collected and cultured for 72 h to the expanded blastocyst stage and similarly evaluated. Embryos were placed in medium containing monoclonal antibodies to H-Y antigen followed by incubation in medium containing 1/10 (v/v) fluorescein isothiocyanate conjugated goat anti-mouse IgM Fc spe...
Murakami Y, Nerome K, Yoshioka Y, Mizuno S, Oya A.Growth characteristics of a wide range of influenza A viruses from different mammals and bird species were examined in an established line of canine kidney (MDCK) cells at an ordinary (37 degrees C) and a high temperature (42 degrees C). Although all viruses employed in the present study possessed a capability of replicating at 37 degrees C, virus growth at 42 degrees C showed considerable variation and reflected differences in the natural hosts of the isolates. All reference strains and isolates from bird species grew well in the MDCK cells maintained at 42 degrees C, but human viruses did no...
Yang JM, Woods WE, Weckman TJ, Wood TW, Chang SL, Blake JW, Tobin T.1. Drug administration studies using diisopropylamine dichloroacetate (DADA) and diisopropylamine (DIPA) were conducted in Thoroughbred and Standardbred horses to assess physiological effects and develop detection methods. 2. Four horses received 0.08 mg DADA/kg body wt and showed no changes in heart and respiratory rates or body temperature as measured over a 1-hr period after administration. A transient diuretic effect was found to occur in 2 mares dosed with 0.80 mg DADA/kg body wt. 3. A qualitative detection method using thin-layer chromatography was developed to detect DIPA, the major met...
Kono Y.The antigenic structure of 16 viruses isolated from four horses which were inoculated with a clone of equine infectious anemia (EIA) virus was compared by the neutralization test. The antigenic structure of viruses isolated after development of neutralizing antibody differed from virus to virus. Back mutation of the antigenic structure was also demonstrated by serial passage of the virus in horses. These results suggest that EIA virus is subject to multidirectional antigenic variation. The possibility that the variants originated in the heterologous virus population in the inoculum seems to be...
Hussain KA, Issel CJ, Schnorr KL, Rwambo PM, West M, Montelaro RC.Monoclonal antibodies (MCAbs) were used to dissect the antigenic sites of the surface glycoproteins of the prototype cell-adapted Wyoming strain of equine infectious anemia virus (EIAV). Serologic reactivities of these MCAbs were determined by ELISA, additive ELISA, competitive ELISA, and Western blot assays. The results indicated that antigenic reactivity of gp90 was localized on at least four distinct epitopes, two of which were important in neutralization. Our studies also revealed that these epitopes were localized on overlapping antigenic sites on gp90. On the other hand, only two distinc...
Jones I, Madej A.Microtitre plates were coated with antiserum against oestradiol-17 beta-6-(O-carboxymethyl)-oxime bovine serum albumin raised in sheep. The plasma samples (0.2-1.0 ml) were extracted with peroxide-free diethyl ether prepared daily by treatment with Al2O3. The enzyme conjugate was prepared by coupling oestradiol-17 beta-6-(O-carboxymethyl)-oxime to horse-radish peroxidase. The conjugate was chromatographed on a Sephadex G-25 column. The standard curve ranged from 0.37 to 18.40 fmol/well of oestradiol-17 beta. The amount of oestradiol-17 beta causing a 50% reduction of maximum binding was 4.4 fm...
Meyers KM, Menard M, Wardrop KJ.This is a review of equine hemostasis and is divided into three sections. The initial portion describes the normal hemostatic system and includes platelet function, coagulation, fibrinolysis and control processes. The second phase is devoted to laboratory tests of hemostasis, and the last section provides information on specific alterations.
López ML, de Souza W, Bustos-Obregón E.The structure, relative density, and distribution of anionic sites on the surface of epididymal and ejaculated spermatozoa were studied using polycationic ferritin (CF), colloidal iron hydroxide (CIH), various enzymatic treatments, methylation, and de-acetylation. Macro-molecules containing sugar residues, probably sialic acid, are part of the sperm membrane and show a characteristic distribution and density that is dependent of the sperm region and of its origin. Unlike the spermatozoa of other eutheria examined, the exposure of the stallion spermatozoa to neuraminidase treatment did not prod...
Ricketts SW.The clinician may use the clinical pathology laboratory as a valuable aid to diagnosis and management, for the assessment of response to treatment, and in preventive medicine programs. Each "link in the chain," that is, sample selection, collection, handling, analysis, result reporting, and interpretation must be carefully and efficiently managed, using an informed combination of art and science, to provide a useful endpoint. This general introduction precedes more specific and detailed articles.
Winston S, Fiscus S, Hesterberg L, Matsushita T, Mildbrand M, Porter J, Teramoto Y.The development of three separate rapid ELISAs for detecting antibodies in host serum to three different viruses is described. These include: 1. A direct antigen assay using enzyme labelled anti-canine Ig for detecting antibodies to canine parvovirus, 2. A competitive ELISA using a feline infectious peritonitis virus-specific monoclonal antibody labelled with enzyme, and 3. A competitive ELISA using an equine infectious anemia virus-specific monoclonal antibody and enzyme labelled antigen, p. 26. The utility and benefits of each of the three approaches is emphasized.
Kohn CW, Chew DJ.Laboratory evaluation of renal function in horses has advanced dramatically in the last 10 years largely as a result of the interest generated by the creative approach to diagnostic indices taken by Brobst, Traver, Coffman, and others. Some methods of assessing renal function discussed here are clearly outside the scope of a practice environment but are available in referral hospitals for use in difficult or unusual cases. Other methods described, such as calculation of fractional excretions and urine to serum creatinine ratios, are accessible and readily interpreted by the veterinary practiti...
Stadler P.The anterior enteritis syndrome in the horse is reviewed with reference to the aetiology, pathogenesis, clinical findings, laboratory findings, diagnosis, treatment, prognosis and post mortem findings.
Engelking LR, Paradis MR.This article addresses clinical problems that present in equine liver disease. It also discusses the variety of laboratory tests available to the clinician that can differentiate the type and degree of liver dysfunction. This is followed by a more specific discussion regarding unique features of equine bilirubin and bile acid metabolism.
Sweeney RW.Malassimilation should be suspected in horses with weight loss in spite of a good appetite. Malassimilation is usually confirmed with oral glucose or D-xylose absorption tests, whereas the oral lactose tolerance test can be used to evaluate lactase deficiency in foals. Once malassimilation is confirmed, other diagnostic tests such as abdominocentesis, rectal mucosal biopsy, or exploratory laparotomy with intestinal biopsies may determine the etiology of malassimilation.
Cowell RL, Tyler RD, Clinkenbeard KD, MacAllister CG.This article discusses collection, slide preparation, culture technique, fluid analysis and evaluation, and cytologic evaluation of peritoneal and pleural effusions. The morphologic characteristics of various effusions are described, and the physical characteristics (volume, color, turbidity) of effusions are discussed. An algorithm for classifying effusions as transudates, modified transudates, or exudates is included, and each category is discussed.
Tyler RD, Cowell RL, Clinkenbeard KD, MacAllister CG.Normal reference ranges and pertinent background information on equine hematology are presented and briefly discussed. Diagnostic interpretation of hematologic data is discussed and three diagnostic algorithms and two diagnostic tables are provided to facilitate the use of the presented information for diagnosis. Two cases are presented and the information presented in the article is used to interpret the case data.
Craig AM, Blythe LL, Appell LH, Slizeski ML.Dimethyl sulfoxide (DMSO) had been postulated to be a 'masking agent' when used concurrently with therapeutic or prohibited drugs in racing animals. Eight drugs (flunixin, furosemide, caffeine, apomorphine, phenylbutazone, lidocaine, cocaine, and acepromazine maleate) were administered to six horses singly and with concurrent intravenous DMSO. Urine samples were analyzed for the presence of the drugs and/or their metabolites by thin layer chromatography. Direct comparison of thin layer chromatograms of extracts of positive urine samples with and without DMSO verified that DMSO did not interfer...
Ammer H, Henschen A.The major stallion protamine was isolated from sperm cell nuclei by extraction with 6M guanidine/5% mercaptoethanol, alkylation with 4-vinylpyridine and subsequent reversed-phase high-performance liquid chromatography. The primary structure of stallion protamine was determined by N-terminal sequencing of the intact protein and of the fragments obtained from thermolysin cleavage of the S-pyridylethylated and from endoproteinase Lys-C cleavage of the S-aminoethylated protein. Stallion protamine consists of 49 amino-acid residues and shows 49% identity with all other sequenced mammalian type 1 pr...
Labadia A, Rivera L, Costa G, García-Sacristán A.The presence of both alpha and beta adrenergic receptors in the caudal third ureter of the horse were studied in vitro under isometric conditions using adrenergic agonist and antagonist drugs. Isoprenaline and the beta 2- stimulating agent, salbutamol, elicited relaxation of the ureter smooth muscle strips. The responses were not affected by the beta 1- blocking agent, practolol, but were totally abolished by propranolol and the beta 2- blocking agent, butoxamine. The stimulation of alpha-adrenergic receptors with noradrenaline and phenylephrine evoked a contractile effect which was totally in...
Satorov SS, Orzuev MI.Staphylococci occur in donkeys more frequently than in other animals, and only from donkeys coagulase-negative staphylococci, characteristic of humans (S. hominis, S. capitis, S. cohnii), were isolated. Least frequently staphylococcal carrier state was registered in cats; in these animals only coagulase-negative strains were found to occur. From 30 donkeys coagulase-positive staphylococci belonging to 47 S. aureus strains were isolated. These strains differed from known ecological variants in their biological properties, thus suggesting the existence of S. aureus ecovar specific for donkeys. T...
Stewart F, Thomson JA, Leigh SE, Warwick JM.Several cDNA clones corresponding to mRNA for the alpha-subunit of the horse (Equus caballus) pituitary and placental (chorionic) gonadotrophic hormones have been isolated and sequenced. Polyadenylated mRNA was purified from horse pituitary glands (the source of FSH and LH) and horse placental tissues (the source of chorionic gonadotrophin; CG). The mRNA preparations were characterized by in-vitro translation and Northern hybridization techniques using human and ovine gonadotrophin cDNA clones as probes. Complementary DNA libraries were created from the pituitary and placental mRNAs and a huma...
Houle JJ, Hoffmann EM.A passive hemolysis assay system was developed which permitted comparisons of the hemolytic activities of complement (C) from six species. This system employs a single antigen and an antiserum raised in one species. Thus, variations resulting from different target antigens and those inherent in using antibodies (of different affinities and isotypes) raised in a variety of species were minimized. Of the erythrocytes (E) examined, those from horses and guinea pigs were most susceptible to lysis, and either would be suitable, as a tentative choice, for measuring C activity of a previously unstudi...
Ignatchenko AP, Bogomaz VI, Tugaĭ VA, Chuĭko AA.Biospecific sorbents for affinity chromatography of proteolytic enzymes have been synthesized by attaching cyclopeptide antibiotic gramicidin S to organo-silica supports. It is shown possible to attach gramicidin S to the organo-silica supports using glutaric aldehyde, p-benzoquinone, soluble and insoluble carbodiimides. The sorbents prepared by these methods were successfully applied for the purification of the crude pepsin from horse gastric juice and proteolytic complex produced by Acremonium chrysogenum.
Nishita T, Matsushita H, Kai M.The location of carbonic anhydrase III (CA-III) in frozen sections of biopsies of Thoroughbred horse skeletal muscle was studied. Fibre types were determined by ATP-ase and succinate dehydrogenase staining. CA-III isozyme was detected using a peroxidase conjugated anti-CA-III antibody. CA-III was found to be localised in slow twitch oxidative fibres (ST), but was also present in fast twitch oxidative (FTH) fibres in small amounts. Fast twitch glycolytic (FT) fibres were stained lightly compared with control sections. The concentrations of CA-III in muscle and liver were 70 micrograms/mg protei...
Vitello LB, Erman JE.The binding of horse heart cytochrome c to yeast cytochrome c peroxidase in which the heme group was replaced by protoporphyrin IX was determined by a fluorescence quenching technique. The association between ferricytochrome c and cytochrome c peroxidase was investigated at pH 6.0 in cacodylate/KNO3 buffers. Ionic strength was varied between 3.5 mM and 1.0 M. No binding occurs at 1.0 M ionic strength although there was a substantial decrease in fluorescence intensity due to the inner filter effect. After correcting for the inner filter effect, significant quenching of porphyrin cytochrome c pe...
Pfeiffer CJ, Murray MJ, Fainter L.The granular columnar epithelial cell of the equine dorsal large colon has been studied by transmission electron microscopy and X-ray microanalysis. Particular attention was focused on nuclear bodies commonly observed as central clusters of spherical, electron-dense inclusions within the nucleus. Ultrastructural morphology as well as X-ray microanalysis spectra reveal great similarity between colonic nuclear bodies and the numerous small granules of the apical cytoplasm in these epithelial cells. X-ray spectra of these cells were distinct from those of goblet cell mucous granules and mast cell...
Weiser MG.Using a single channel electronic cell counter and attached particle size analyzer, leukocyte size distribution histograms were determined on canine, feline, bovine, and equine blood diluted with chloride-based diluent and treated with a conventional stromatolysin. Histograms were usually unimodal, but a few were bimodal. Mean values for mean lysed leukocyte particle volume were 49.2, 51.1, 55.4, and 65.0 fl for canine, feline, equine, and bovine blood, respectively. From inspection of histograms, a lower threshold of 30 fl referenced to latex spheres was interpreted to be appropriate for coun...
Fu ZF, Denby L, Lien DH, Robinson AJ.An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies against equine herpesvirus type 2 (EHV-2) in equine sera. The optimal conditions of antigen concentration, and serum and conjugate dilutions were established by chequerboard titrations. When the standard ELISA test was used for titration of test sera, it was found to give titres approximately 1500 times higher than those obtained in the virus neutralization (VN) test, and a correlation coefficient of 0.815 was obtained between these two tests on 42 equine sera. All the positive serum samples by ...
Babasyan S, Rollins A, Wagner B.Interleukin-1β (IL-1β) is one of the key mediators of inflammation during innate immune responses. Mature bioactive IL-1β mediates essential host defense mechanisms but also has a mechanistic role in several autoinflammatory and degenerative diseases. In horses, specific and sensitive assays for IL-1β are crucial for immunological research on inflammatory processes and diseases. In this article, we describe the development of four monoclonal antibodies (mAbs) against equine IL-1β. The specificity of the new IL-1β mAbs was confirmed using a panel of equine recombinant cytokines and chemok...
Tikmehdash HT, Dehnad A, Mosavari N, Naghili Hokmabadi B, Mahmazi S.Glanders caused by Burkholderia mallei is one of the most dangerous zoonotic diseases in solipeds. Clinical diagnosis of this disease in its early stages in horses, is difficult. This study investigated serological and molecular identification of B. mallei in East Azerbaijan province. In the third and fourth quarters of 2020, throughout 2021, and in the first and second quarters of 2022, the complement fixation test (CFT) was performed on 350 horses. The malleination was used to confirm the positive CFT cases. Blood samples were taken for culture and for preparing serums to perform the enzyme-...
Woo PCY, Mheiri FA, Cavalleri J, Joseph S, Tang JYM, Joseph M, Tsang CC, Lau SKP, Wernery U.Epizootic lymphangitis (EL) is a highly prevalent and contagious infectious disease affecting horses in many parts of Ethiopia caused by Histoplasma capsulatum sensu lato ('var. farciminosum'). In this study, 12 suspected isolates of H. capsulatum sensu lato or yeasts unidentified by conventional biochemical tests isolated from Ethiopian horses with EL were characterised by ITS sequencing. Six of the 12 isolates were identified to be members of H. capsulatum sensu lato and the other six were Pichia kudriavzevii (synonym: Candida krusei) (n = 3), Trichosporon asahii (n = 1), Geotrichum silv...
Lacante SA, Jiang C, Mustamir AA, Mizuno T, Bi X, Syafruddin D, Tokoro M., a human intestinal protozoan parasite of the diplomonad group, has been overlooked because of its commensal features; therefore, molecular studies on this parasite are limited. To address this gap, we designed a molecular screening protocol using polymerase chain reaction (PCR) and DNA sequencing targeting the 18S small subunit ribosomal RNA gene and applied this screening method to the molecular epidemiological analysis of spp. in humans and various livestock. We validated our methodology using stool samples collected from 215 humans and 270 animal hosts (buffaloes, pigs, dogs, goats, hors...
Lawrence KE, Gedye K, Carvalho L, Wang B, Fermin LM, Pomroy WE.To determine whether evidence for infection with (Ikeda) could be identified in samples of commercial red deer , horses, and working farm dogs in New Zealand. Unassigned: Blood samples were collected during October and November 2019 from a convenience sample of red deer (n = 57) at slaughter. Equine blood samples (n = 50) were convenience-sampled from those submitted to a veterinary pathology laboratory for routine testing in January 2020. Blood samples, collected for a previous study from a convenience sample of Huntaway dogs (n = 115) from rural regions throughout the North and ...
Ochi A, Toya Y, Sengoku M, Tsuchiya S, Kishi D, Ueno T.Equine piroplasmosis (EP) is a protozoal disease affecting equids, caused by and . EP is conventionally diagnosed using microscopic, molecular, and/or serological methods, which are time-consuming. Consequently, there is a need for faster testing methods. In this study, we evaluated the application of the Sysmex XN-31 automated hematology analyzer, originally a rapid test for detecting malaria in humans, for the diagnosis of EP. The cultured parasites were measured using the XN-31 that had been customized for horse blood samples (XN-31m). The following parameters were evaluated: limit of dete...
Valera M, Karlau A, Anaya G, Bugno-Poniewierska M, Molina A, Encina A, Azor PJ, Demyda-Peyrás S.Sex chromosomal abnormalities are a well-established cause of reproductive failure in domestic horses. Because of its difficult diagnosis, the Pura Raza Español breeding program established a routine screening for chromosomal abnormalities in all the horses prior to enrolling in the studbook. This genomic procedure combines an initial assessment based on the results from Short Tandem Repeat (STR) parentage testing followed by a Single-Nucleotide Polymorphism (SNP) based copy number aberration (CNA) confirmative analysis in positive cases. Using this methodology, we identified five new individ...
Ali YH, Mohieddeen TAG, Abdellatif MM, Ahmed BM, Saeed IK, Attaalfadeel HM, Ali AA.Rabies is endemic in Sudan with continuing outbreaks occurring annually, the most common animals affected are dogs, followed by goats and equids. This work focused on equid rabies, to elucidate the current situation of the disease through analysis of reports of equid rabies outbreaks in Sudan during 2010-2022 supported by laboratory confirmation of the disease. During the study period, 66 animals were affected during 35 equid rabies outbreaks. The highest incidences were found in Al Gezira (30.3%), followed by Darfur (24.2%) and Kordofan (15.2%). The highest incidence rate was observed during ...
Bayless RL, Cooper BL, Sheats MK.Colic is a common and potentially life-threatening condition in horses; in many cases, it remains challenging for clinicians to determine the cause, appropriate treatment, and prognosis. One approach that could improve patient care and outcomes is identification of novel diagnostic and prognostic biomarkers. Plasma cell-free DNA (cfDNA) is a biomarker that shows promise for characterizing disease severity and predicting survival in humans with acute abdominal pain or requiring emergency abdominal surgery. In horses, we recently determined that extracted plasma cfDNA concentrations are elevated...
Loup B, André F, Leuenberger N, Marchand A, Barnabé A, Delcourt V, Garcia P, Popot MA, Bailly-Chouriberry L.Detection and monitoring of biomarkers related to doping is particularly suitable for the development of analytical strategies dedicated to indirect detection of banned substances. Previous studies in horses have already allowed the investigation of transcriptomic biomarkers in equine blood associated with reGH and rHuEPO administrations. Our most recent developments continue to focus on the discovery and monitoring of transcriptomic biomarkers for the control of ESAs, and a collaborative study with WADA-accredited doping control laboratories has recently been initiated to conduct a pilot stud...
Buschmann E, Van Steenkiste G, Duytschaever M, Segers P, Ibrahim L, van Loon G, Decloedt A.Radiofrequency ablation is a promising technique for arrhythmia treatment in horses. Due to the thicker myocardial wall and higher blood flow in horses, it is unknown if conventional radiofrequency settings used in human medicine can be extrapolated to horses. The study aim is to describe the effect of ablation settings on lesion dimensions in equine myocardium. To study species dependent effects, results were compared to swine myocardium. Right ventricular and right and left atrial equine myocardium and right ventricular swine myocardium were suspended in a bath with circulating isotonic sali...
Velineni S, Schiltz P, Chang KH, Peng YM, Cowles B.Abnormal blood glucose (BG) levels often seen in critically ill horses are significantly associated with adverse patient outcomes and increased mortality. Rapid and accurate BG monitoring is now considered an essential component of evidence-based equine practice and can provide critical information quickly for treatment. Although several point-of-care (POC) BG monitoring hand-held devices are commercially available for veterinary use, none contains a unique algorithm validated for use in horses. The AlphaTrak 3 (AT3) BG monitoring system is a first-of-its-kind device with an equine-specific al...
Lehman ML, Domenig O, Ames MK, Morgan JM.Furosemide, a commonly used diuretic, activates the renin-angiotensin-aldosterone system (RAAS) in other species. Little is known about RAAS peptide activation in horses. Objective: To evaluate equilibrium analysis as a practical method for RAAS quantification in horses and describe the RAAS response to a single dose of furosemide. We hypothesize that furosemide would cause transient increase in RAAS peptides in horses. Methods: 14 healthy adult thoroughbreds from a university teaching herd. Methods: Horses received either furosemide (1 mg/kg IV) or saline IV in a crossover study design. Pro...
Craig NM, Munguia NS, Trujillo AD, Chan AM, Wilkes R, Dorr M, Marsella R.This study investigated the effects of recombinant equine IL-31 (eIL-31) in vivo and in vitro. Methods: Equine IL-31 mRNA sequences were verified by sequencing. Recombinant eIL-31 was produced using mammalian and bacterial expression systems. From November 2019 through February 2021, 12 normal horses, 6 to 10 years old with no history or clinical signs consistent with allergic skin disease, were injected ID with eIL-31 and saline in 2 challenge studies. Pruritus-associated behaviors were recorded for a minimum of 15 minutes preinjection and 4 hours postinjection. Adherent monocytes from 3 prur...
Brito LFC, Felix MR, Linardi RL, Martinez de Andino EV, Balamurugan NS, Hernández-Avilés C, Hinrichs K.Intracytoplasmic sperm injection (ICSI) is a valuable assisted reproduction technology in clinical practice, especially when semen availability is limited. Since the number of sperm required per ICSI cycle is much less than the number of sperm available in a standard straw of frozen semen, refreezing semen at lower sperm concentrations could yield multiple straws for ICSI use. However, there is little data on the effect of sperm refreezing on ICSI outcomes, especially on the effect of extender used for refreezing. The objective of the present study was to evaluate the effect of refreezing exte...
Pooja , Thapa N, Rani R, Shanmugasundaram K, Jhandai P, Rakshita , Bhattacharya TK, Singha H.Immunological aspects of B. mallei infection were rarely studied in natural cases of equines. The present study was conducted to determine IgG, IgM, IgA and IgG (T) titre against recombinant Hcp1, TssA and TssB proteins and PilA-Hcp1-TssN-BipD and BpaB-BpaC- BMAA0553 chimeras and cytokine responses in glanders affected equine serum. Methods: The study was conducted on serum samples collected from 151 glanders positive equines which include horses (n = 134), mules (n = 16), and donkeys (n = 01). The IgM, IgG, IgA and IgG (T) titre were determined against recombinant antigens by indirect E...
Du M, Li X, Bayinnamula , Wang N, Liu Y, Zhang L, Zhao Y, Dugarjaviin M.An important method for preserving equine germplasm is the cryopreservation of equine oocytes. Due to its ease, rapidity and affordability, vitrification freezing has taken over as the primary method of horse oocyte cryopreservation. The vitrification cryoprotectants utilized in this investigation were Ethylene glycol (E), Dimethyl sulfoxide (D), Sucrose (S), and Ficoll (F). According to the oocyte volume alteration, the treatment time was 39 s in equilibrium solution ED10 (10 % EG + 10 % DMSO), 32 s in equilibrium solution ED15 (15 % EG + 15 % DMSO), while 20 s in equilibrium so...
Scupham AJ, Tong C.In 2018, a new virus, named equine parvovirus-hepatitis (EqPV-H), was discovered in a biologic product that had been administered to horses that subsequently developed clinical signs of equine serum hepatitis (Theiler disease). Further correlation of the virus with the disease sparked federal requirements that all equine biologics be free of EqPV-H. The initial quantitative real-time PCR (qPCR) test for EqPV-H has proved to be sensitive to co-extracted PCR inhibitors in template nucleic acids, causing false-negative results. We investigated the use of digital PCR (dPCR) as a more robust test. ...
Ohnuma K, Hirano-Kodaira M, Bannai M, Shimizu Y, Yamada M, Kinoshita K, Ngai-Wa Leung G, Ishii H.The abuse of prohibited peptide-based drugs with a broad spectrum of chemical characteristics poses a significant concern for the horseracing industry. Recently, there has been a notable increase in positive cases of small-peptide drugs reported in equine and canine sports. In addition to small peptides, large peptides (over 2 kDa) with structural diversity have also entered the market in increasing numbers as drugs for humans and livestock. However, the simultaneous analysis of both small- and large-peptide-based drugs is still challenging. In this study, a screening method was developed to c...
Vokes JR, Gedye KR, Lovett AL, de Kantzow MC, Shan R, Steel CM, Sykes BW.Gastrin is an important hormone involved in gastric acid secretion. Despite its importance, validated methods other than radioimmunoassay (RIA) to assess serum gastrin concentrations in horses are lacking. This study aims to determine the agreement between ELISA and RIA in quantifying equine serum gastrin concentrations. Serum gastrin concentrations were quantified using two ELISA kits and RIA. Samples (196) from 14 horses at different time points were analyzed using one ELISA kit and RIA, selected samples (7) were analyzed using a second ELISA kit, and the correlation between methods was calc...
Längerer L, Schuler G, Büttner K, Wehrend A.The determination of progesterone from mares' serum plays a decisive role in diagnosing estrus cycle disorders or luteal insufficiency. To date, no measurement methods are available for rapid quantitative diagnosis of serum progesterone in the mare that would allow results to be available within a two-hour time frame. The present study will evaluate a commercial enzyme-linked fluorescent assay, the mini VIDAS device (bioMérieux, Nürtingen, Germany). Serum was prepared from the blood samples of one hundred and seven mares, divided into two aliquots, and stored at -20°C. Subsequently, compara...
Bishop RC, Shanthappa N, Connolly SL, Wilkins PA, McCoy AM.To establish the reference interval (RI) of fecal calprotectin (fCP) and fCP:protein ratio in the feces of healthy horses and demonstrate preliminary clinical utility for the quantification of intestinal inflammation. Methods: Feces were collected from healthy horses (n = 103) and horses with colic (n = 15) or colitis (n = 13). Feces were suspended in buffer to create fecal supernatant. Fecal calprotectin concentration was determined by ELISA, fecal total protein concentration was determined by bicinchoninic acid assay, and the fCP:protein ratio was calculated. Reference intervals for fCP and ...
Koziy RV, Katselis GS, Yoshimura S, Simko E, Bracamonte JL.Prompt diagnosis of equine septic arthritis is crucial for successful treatment. Serum amyloid A (SAA) has been suggested as a reliable biomarker. However, we previously found that synovial fluid SAA increases in nonaffected joints of horses with septic arthritis. We hypothesized that systemic SAA may leak into the nonaffected joints. If this is the case, we also hypothesized that locally produced joint SAA isoforms may be better candidates for septic arthritis biomarkers. Thus, our objectives were 1) to evaluate the temporal kinetics of systemic and synovial fluid SAA in horses with septic ar...
Wong JKY, Choi TLS, Wong COL, Curl P, Wan TSM, Ho ENM.Methylsulfonylmethane (MSM), also known as dimethyl sulfone, is a naturally occurring sulphur-containing compound that can be found in plants, animals and humans. MSM can also be a metabolite of dimethyl sulfoxide (DMSO). Due to their anti-inflammatory and analgesic effects, both MSM and DMSO are prohibited substances in horseracing. As both substances are naturally occurring, their misuse in horses is controlled by International Residue Limits (IRL) of 1200 and 15 μg/mL, respectively, in horse urine as established by the International Federation of Horseracing Authorities. The elimination ...
Valderrama-Martinez C, Packham A, Zheng S, Smith W, Plancarte M, Aleman M.Evaluating antibody titers for Sarcocystis neurona for the diagnosis of equine protozoal myeloencephalitis from serum samples is a common practice. However, ensuring timely and proper refrigeration is not always possible. Objective: To evaluate immunofluorescent antibody (IFA) titers for S. neurona from serum samples stored at room temperature and 4°C. Methods: Twenty-two serum samples. Methods: Prospective longitudinal study. Two serum aliquots of 1 mL each were stored at room temperature (20-23.3°C) and 4°C. The unrefrigerated aliquot was immediately tested for IFA titers. Both aliquots...
Broeckl CV, Hiereth S, Straubinger RK.Diagnosis of equine Lyme borreliosis (LB), an infection caused by members of the Borrelia burgdorferi sensu lato complex (Bbsl), is challenging due to the nonspecific clinical signs of the disease and due to the variety of non-standardized serological tests. Specific vaccine-induced antibodies against LB, providing an effective protection against the infection, complicate the issue further. The standard for the detection of specific antibodies against Bbsl is a two-tier test system based on an enzyme-linked immunosorbent assay (ELISA) or indirect fluorescent antibody test (IFA) for antibody sc...
Palomino Lago E, Ross AKC, McClellan A, Guest DJ.Bone fractures are a significant problem in Thoroughbred racehorses. The risk of fracture is influenced by both genetic and environmental factors. To determine the biological processes that are affected in genetically susceptible horses, we utilised polygenic risk scoring to establish induced pluripotent stem cells (iPSCs) from horses at high and low genetic risk. RNA-sequencing on iPSC-derived osteoblasts revealed 112 genes that were significantly differentially expressed. Forty-three of these genes have known roles in bone, 27 are not yet annotated in the equine genome and 42 currently have ...
Königová A, Babják M, Kuzmina TA, Burcáková Ľ, Syrota Y, Várady M.The study aimed to conduct a survey on the occurrence of benzimidazole (BZ) resistance in strongyles by in vitro egg hatch test (EHT) and larval development test (LDT) and to identify the effective indicators of early resistance detection on horse farms with associated risk factors analysis appraisal. In total, 203 horses from 8 farms underwent the fecal sampling of which 77 horses were selected for in vitro testing. Simultaneously, 18 horses were chosen to analyse the results of in vitro tests compared to the in vivo fecal egg count reduction test (FECRT). The EHT indicated the presence of re...
Chen JW, Stefanovski D, Haughan J, Jiang Z, Soma LR, Robinson MA.Interleukin 1 beta (IL-1β) and IL-1 receptor antagonist (IL-1RA) are both upregulated following traumatic injury. As IL-1RA blocks inflammatory signaling by IL-1β, overexpression of IL-1β relative to IL-1RA may drive inflammatory diseases. As such, determination of the relationship between IL-1β to IL-1RA expression levels in horses may provide insight into disease states or serve as a therapeutic readout of response to medical interventions. As techniques to detect plasma concentrations of IL-1β and IL-1RA in horses lack sensitivity, we developed and validated novel enzyme-linked immunos...
Nemoto M, Kawanishi N, Kambayashi Y, Bannai H, Yamanaka T, Tsujimura K.Equine influenza virus (EIV) can be transmitted by inhalation of aerosolized droplets, direct contact, and contaminated fomites. However, to our knowledge, there are no reports of the recovery of EIV from the air surrounding infected horses. Here, we evaluated whether EIV can be recovered from the air in the stalls of experimentally infected horses by using an air sampler. Furthermore, we examined whether rapid molecular test kits with reaction times of less than 30 min can detect EIV from air samples for potential field application. Two horses kept in individual stalls were experimentally i...
